RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (SINA)

ABSTRACT

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of HBV gene expression and/or activity, and/or modulate a HBV gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against HBV gene expression.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/844,793filed on Dec. 18, 2017, now U.S. Pat. No. 10,407,682, issued on Sep. 10,2019, which is a continuation of application Ser. No. 15/251,155, filedon Aug. 30, 2016, now U.S. Pat. No. 9,879,262, issued on Jan. 30, 2018,which is a continuation of application Ser. No. 14/682,618, filed onApr. 9, 2015, now U.S. Pat. No. 9,464,290, issued on Oct. 11, 2016,which is a continuation of application Ser. No. 13/817,152, filed onFeb. 15, 2013, now U.S. Pat. No. 9,029,341, issued on May 12, 2015,which is a 35 U.S.C. § 371 National Stage filing of InternationalApplication No. PCT/US2011/047512, filed on Aug. 12, 2011, which claimsthe benefit of and priority to U.S. Provisional Patent Application Ser.No. 61/374,555, filed on Aug. 17, 2010. The entire contents of each ofthe foregoing patent applications are incorporated herein by reference.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file 930385 407C4 SEQUENCELISTING.txt, creation date of Aug. 5, 2019 and a size of 455,797 bytes.This sequence listing submitted via EFS-Web is part of the specificationand is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Hepatitis B is an infectious disease of the liver caused by theHepatitis B virus (HBV). The illness can be acute causing liverinflammation, vomiting, jaundice and in some rare instances of severefulminant disease, death. The majority of infections result in chronicillness that can be either asymptomatic or resulting in chronic liverinflammation leading to cirrhosis of the liver and an increasedincidence in the development of hepatocellular carcinoma (HCC).

HBV infection is a global problem with approximately >350 million peopleworld wide chronically infected and 600,000 die each year fromHBV-related liver disease or HCC. The disease has caused epidemics inAsia and Africa and is endemic in China. Transmission of HBV is viainfectious blood or body fluids. There are currently vaccines for theprevention of HBV infection. However, the vaccine is prophylatic andcannot be used to treat already infected patients.

There are several approved chemotherapeutic treatments for chronichepatitis B (lamivudine, adefovir, entacavir and telbivudine) thatprevent replication of HBV by blocking the action of the HBV polymerase.None of the treatments result in complete clearance of the virus andthese treatment result in drug-resistant HBV variants developing afterprolonged treatment. Hepatitis B can also be treated with pegylatedInterferon-alpha2a but this treatment is associated with severe sideeffects and is not effective in all patients.

HBV is a member of the Hepadanvirus family and is divided into 4 majorserotyoes (adr, adw, ayr, ayw) based on antigenic epitopes. The virus isalso classed into eight genotypes (A-H based on genomic sequence.Genotype A is common in the Americas, Africa, India and Western Europe.Genotype B and C are found in Asia and the US. Genotype D is most commonin Southern Europe and India. Genotype E is found in Western andSouthern Africa. Genotype F and H are commonly found in Central andSouthern America. Genotype G is commonly found in Europe and the U.S.

HBV genome consists of a circular strand of DNA that is partially doublestranded. The genome is ˜3.3 Kb in length. It encodes 4 known genes (C,X, P and S). HBV is one of the few DNA viruses that utilize reversetranscriptase in the replication process. HBV replication involvesmultiples stages including entry, uncoating and transport of the virusgenome to the nucleus. Initially, replication of the HBV genome involvesthe generation of an RNA intermediate that is then reverse transcribedto produce the DNA viral genome.

Since current therapies are limited due to their ineffectiveness,serious side effects or due to the generation of drug resistantvariants, there is a clinical need for the development of new therapiesto treat HBV infection.

Alteration of viral gene expression, specifically HBV gene expression,through RNA interference (hereinafter “RNAi”) is one approach formeeting this need. RNAi is induced by short single-stranded RNA(“ssRNA”) or double-stranded RNA (“dsRNA”) molecules. The short dsRNAmolecules, called “short interfering nucleic acids (“siNA”)” or “shortinterfering RNA” or “siRNA” or “RNAi inhibitors” silence the expressionof messenger RNAs (“mRNAs”) that share sequence homology to the siNA.This can occur via cleavage of the mRNA mediated by an endonucleasecomplex containing a siNA, commonly referred to as an RNA-inducedsilencing complex (RISC). Cleavage of the target RNA typically takesplace in the middle of the region complementary to the guide sequence ofthe siNA duplex (Elbashir et al., 2001, Genes Dev., 15:188). Inaddition, RNA interference can also involve small RNA (e.g., micro-RNAor miRNA) mediated gene silencing, presumably through cellularmechanisms that either inhibit translation or that regulate chromatinstructure and thereby prevent transcription of target gene sequences(see for example Allshire, 2002, Science, 297:1818-1819; Volpe et al.,2002, Science, 297:1833-1837; Jenuwein, 2002, Science, 297:2215-2218;and Hall et al., 2002, Science, 297:2232-2237).

Several studies have attempted to use RNAi for the treatment of HBV andthis approach has been comprehensively reviewed in RNAI for treatingHepatitis B Viral Infection, Chen, Y. et al., Pharmaceutical Research,2008 Vol. 25, No. 1, pgs 72-86. Yet, as noted in the above referencetransfection of a single siRNA often fails to provide adequate genesilencing. Id. Thus, despite significant advances in the field of RNAi,there remains a need for agents that can effectively inhibit HBV geneexpression and that can treat disease associated with HBV expressionsuch as liver disease and cancer.

SUMMARY OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat respond to the modulation of the HBV gene expression using novelshort interfering nucleic acid (siNA) molecules to modulate HBVexpression.

The present invention provides compounds, compositions, and methodsuseful for modulating the expression of HBV genes and for treatinghepatitis B and such conditions that result from hepatitis B infectionby RNA interference (RNAi) using small nucleic acid molecules.

In particular, the instant invention features small nucleic acidmolecules, i.e., short interfering nucleic acid (siNA) moleculesincluding, but not limited to, short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA(shRNA) and circular RNA molecules and methods used to modulate theexpression of HBV genes and/or other genes involved in pathways of HBVgene expression and/or activity. The inventors have found that selectionof targets sites from the conserved regions of all known full HBVgenomes resulted in sequences with high potency and efficacy.

In one aspect, the invention provides double-stranded short interferingnucleic acid (siNA) molecules that inhibit the expression of a HBV genein a cell or mammal, wherein the double-stranded siNAs comprise a senseand an antisense stand. The antisense strand comprises a sequence thatis complementary to at least a part of an RNA associated with theexpression of the HBV gene. The sense strand comprises a sequence thatis complementary to the antisense strand. In various embodiments, atleast one strand comprises at least a 15 nucleotide sequence selectedfrom the group of sequences consisting of SEQ ID NOS:1-502. In certainembodiments, the antisense strand comprises at least 15 nucleotideshaving sequence complementarity to a target sequence set forth in Table1a. In other and/or in the same embodiments, the antisense strandcomprises at least a 15 nucleotide sequence of one of the antisensesequences set forth in Table 1b. In some embodiments, the sense strandcomprises at least a 15 nucleotide sequence of a sense strand sequenceas set forth in Table 1b.

In certain embodiments of this aspect of the invention, double-strandedshort interfering nucleic acid (siNA) molecules are provided wherein theantisense stand comprises a modified sequence as set forth in Table 1cthat has sequence complementarity to a target sequence of the invention.In some embodiments, the sense strand also comprises a modified sequenceas set forth in Table 1c.

In certain embodiments, the present invention provides a double-strandedshort interfering nucleic acid (siNA) molecule that modulates theexpression of HBV, wherein the siNA comprises a sense strand and anantisense strand; each strand is independently 15 to 30 nucleotides inlength; and the antisense strand comprises at least 15 nucleotideshaving sequence complementary to any of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′; (SEQ ID NO: 2)5′-GUGGUGGACUUCUCUCAAU-3′; (SEQ ID NO: 3) 5′-GCCGAUCCAUACUGCGGAA-3′; or(SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′.

In some embodiments of the invention, the antisense strand of a siNAmolecule comprises at least a 15 nucleotide sequence of:

(SEQ ID NO: 452) 5′-UGAGAGAAGUCCACCACGA-3′; (SEQ ID NO: 453)5′-AUUGAGAGAAGUCCACCAC-3′; (SEQ ID NO: 454) 5′-UUCCGCAGUAUGGAUCGGC-3′;or (SEQ ID NO: 455) 5′-GUUCCGCAGUAUGGAUCGG-3′.

In some embodiments, the sense strand of a siNA molecule of theinvention comprises at least a 15 nucleotide sequence of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′; (SEQ ID NO: 2)5′-GUGGUGGACUUCUCUCAAU-3′; (SEQ ID NO: 3) 5′-GCCGAUCCAUACUGCGGAA-3′; or(SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′.

In some embodiments, a siNA molecule of the invention comprises any of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′ and (SEQ ID NO: 452)5′-UGAGAGAAGUCCACCACGA-3′; or (SEQ ID NO: 2) 5′-GUGGUGGACUUCUCUCAAU-3′;and (SEQ ID NO: 453) 5′-AUUGAGAGAAGUCCACCAC-3′; or (SEQ ID NO: 3)5′-GCCGAUCCAUACUGCGGAA-3′ and (SEQ ID NO: 454)5′-UUCCGCAGUAUGGAUCGGC-3′; or (SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′;and (SEQ ID NO: 455) 5′-GUUCCGCAGUAUGGAUCGG-3′.

In some embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule comprising any of duplexR-008380648-000E, R-008380783-000R, R-008380665-000N, R-008380645-000D,R-008380408-000R, R-008380633-000 U, R-008380767-000R, R-008380730-000C,R-008380777-000H, R-008380740-000V, R-008380558-000M, R-008380406-000Y,R-008380764-000P, R-008380772-000P, R-008380389-000D, R-008380362-000H,R-008380363-000S, R-008380340-000F, R-008380689-000H, R-008380756-000P,R-008380736-000E, R-008380757-000Y, R-008380753-000N, R-008380737-000N,R-008380734-000M, or R-008380791-000R

In some embodiments, the invention features a composition comprising:

-   -   (a) a double-stranded short interfering nucleic acid (siNA) of        the invention;    -   (b) a cationic lipid compound having any of compound numbers        1-46 or any combination thereof;    -   (c) cholesterol;    -   (d) DSPC; and    -   (e) PEG-DMG.

In some embodiments, a composition of the invention comprises anyCationic Lipid having any of compound numbers 1-46 in the followingmolar ratios:

-   -   Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;    -   Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;    -   Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;    -   Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;    -   Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;    -   Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10;    -   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and    -   Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.

In some embodiments, a composition of the invention comprises(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol,DSPC, and PEG-DMG, having a molar ratio of about 50:30:10:2respectively.

In some embodiments, a composition of the invention further comprises acryoprotectant. In some embodiments, the cryoprotectant is Sucrose,Trehalose, Raffinose, Stachyose, Verbascose, Mannitol, Glucose, Lactose,Maltose, Maltotriose-heptaose, Dextran, Hydroxyethyl Starch, Insulin,Sorbitol, Glycerol, Arginine, Histidine, Lysine, Proline,Dimethylsulfoxide or any combination thereof. In some embodiments, thecryoprotectant is Sucrose. In some embodiments, the cryoprotectant isTrehalose. In some embodiments, the cryoprotectant is a combination ofSucrose and Trehalose.

In some embodiments of the invention, all of the nucleotides of siNAs ofthe invention are unmodified. In other embodiments, one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positionsindependently in either one or both strands of an siNA molecule aremodified. Modifications include nucleic acid sugar modifications, basemodifications, backbone (internucleotide linkage) modifications,non-nucleotide modifications, and/or any combination thereof. In certaininstances, purine and pyrimidine nucleotides are differentiallymodified. For example, purine and pyrimidine nucleotides can bedifferentially modified at the 2′-sugar position (i.e., at least onepurine has a different modification from at least one pyrimidine in thesame or different strand at the 2′-sugar position). In certain instancesthe purines are unmodified in one or both strands, while the pyrimidinesin one or both strands are modified. In certain other instances, thepyrimidines are unmodified in one or both strands, while the purines inone or both strands are modified. In some instances, at least onemodified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, a 2′-deoxynucleotide, or a 2′-O-alkyl nucleotide. In some instances, at least 5 ormore of the pyrimidine nucleotides in one or both stands are either all2′-deoxy-2′-fluoro or all 2′-O-methyl pyrimidine nucleotides. In someinstances, at least 5 or more of the purine nucleotides in one or bothstands are either all 2′-deoxy-2′-fluoro or all 2′-O-methyl purinenucleotides. In certain instances, wherein the siNA molecules compriseone or more modifications as described herein, the nucleotides atpositions 1, 2, and 3 at the 5′ end of the guide (antisense) strand areunmodified.

In certain embodiments, the siNA molecules of the invention have 3′overhangs of one, two, three, or four nucleotide(s) on one or both ofthe strands. In other embodiments, the siNA molecules lack overhangs(i.e., have blunt ends). Preferably, the siNA molecule has 3′ overhangsof two nucleotides on both the sense and antisense strands. Theoverhangs can be modified or unmodified. Examples of modifiednucleotides in the overhangs include, but are not limited to, 2′-O-alkylnucleotides, 2′-deoxy-2′-fluoro nucleotides, locked nucleic acid (LNA)nucleotides, or 2′-deoxy nucleotides. The overhang nucleotides in theantisense strand can comprise nucleotides that are complementary tonucleotides in the HBV target sequence. Likewise, the overhangs in thesense stand can comprise nucleotides that are in the HBV targetsequence. In certain instances, the siNA molecules of the invention havetwo 3′ overhang nucleotides on the antisense stand that are 2′-O-alkyl(e.g., 2′-O-methyl) nucleotides and two 3′ overhang nucleotides on thesense stand that are 2′-deoxy nucleotides. In other instances, the siNAmolecules of the invention have two 3′ overhang nucleotides that are2′-O-alkyl (e.g., 2′-O-methyl) nucleotides on both the antisense standand on the sense stand. In certain embodiments, the 2′-O-alkylnucleotides are 2′-O-methyl uridine nucleotides. In certain instances,the overhangs also comprise one or more phosphorothioate linkagesbetween nucleotides of the overhang.

In some embodiments, the siNA molecules of the invention have caps (alsoreferred to herein as “terminal caps.” The cap can be present at the5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present onboth termini, such as at the 5′ and 3′ termini of the sense strand ofthe siNA.

In some embodiments, the siNA molecules of the invention arephosphorylated at the 5′ end of the antisense strand. The phosphategroup can be a phosphate, a diphosphate or a triphosphate.

The siNA molecules of the invention when double stranded can besymmetric or asymmetric. Each strand of these double stranded siNAsindependently can range in nucleotide length between 3 and 30nucleotides. Generally, each strand of the siNA molecules of theinvention is about 15 to 30 (i.e., about 19, 20, 21, 22, 23 or 24)nucleotides in length.

The siNA molecules of the invention, which are double stranded or have aduplex structure, independently comprise about 3 to about 30 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs. Generally, theduplex structure of siNAs of the invention is between 15 and 30, moregenerally between 18 and 25, yet more generally between 19 and 24, andmost generally between 19 and 21 base pairs in length.

In certain embodiments, double-stranded short interfering nucleic acid(siNA) molecules are provided, wherein the molecule has a sense strandand an antisense strand and comprises formula (A):B—N_(X3)(N)_(X2)—(N)_(X2)B-3′B(N)_(X1)—N_(X4)—[N]_(X5)-5′  (A)

-   -   wherein, the upper strand is the sense strand and the lower        strand is the antisense strand of the double-stranded nucleic        acid molecule; wherein the antisense strand comprises at least a        15 nucleotide sequence of SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID        NO: 454, or SEQ ID NO: 455, and the sense strand comprises a        sequence having complementarity to the antisense strand;    -   each N is independently a nucleotide which is unmodified or        chemically modified or a non-nucleotide;    -   each B is a terminal cap that is present or absent;    -   (N) represents overhanging nucleotides, each of which is        independently unmodified or chemically modified;    -   [N] represents nucleotides that are ribonucleotides;    -   X1 and X2 are independently integers from 0 to 4;    -   X3 is an integer from 15 to 30;    -   X4 is an integer from 9 to 30; and    -   X5 is an integer from 0 to 6, provided that the sum of X4 and X5        is 15-30.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   (a) one or more pyrimidine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   (b) one or more purine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   (c) one or more pyrimidine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof; and    -   (d) one or more purine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides,

The present invention further provides compositions comprising thedouble-stranded nucleic acid molecules described herein with optionallya pharmaceutically acceptable carrier or diluent.

The administration of the composition can be carried out by knownmethods, wherein the nucleic acid is introduced into a desired targetcell in vitro or in vivo.

Commonly used techniques for introduction of the nucleic acid moleculesof the invention into cells, tissues, and organisms include the use ofvarious carrier systems, reagents and vectors. Non-limiting examples ofsuch carrier systems suitable for use in the present invention includeconjugates, nucleic-acid-lipid particles, lipid nanoparticles (LNP),liposomes, lipoplexes, micelles, virosomes, virus like particles (VLP),nucleic acid complexes, and mixtures thereof.

The compositions of the invention can be in the form of an aerosol,dispersion, solution (e.g., an injectable solution), a cream, ointment,tablet, powder, suspension or the like. These compositions may beadministered in any suitable way, e.g. orally, sublingually, buccally,parenterally, nasally, or topically. In some embodiments, thecompositions are aerosolized and delivered via inhalation.

The molecules and compositions of the present invention have utilityover a broad range of therapeutic applications. Accordingly anotheraspect of this invention relates to the use of the compounds andcompositions of the invention in treating a subject. The invention thusprovides a method for treating a subject, such as a human, sufferingfrom a condition which is mediated by the action of HBV, wherein themethod comprises administering to the subject an effective amount of adouble-stranded short interfering nucleic acid (siNA) molecule of theinvention. Thus, the siNA molecules of the invention treat the hepatitisB infection and/or conditions resulting therefrom. In some embodiments,the condition is liver disease. In other embodiments, the condition iscancer.

These and other aspects of the invention will be apparent upon referenceto the following detailed description and attached figures. Moreover, itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Additionally, patents, patent applications, and other documents arecited throughout the specification to describe and more specifically setforth various aspects of this invention. Each of these references citedherein is hereby incorporated by reference in its entirety, includingthe drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms thatrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 2 shows non-limiting examples of chemically modified siNAconstructs of the present invention using a generalized structure of arepresentative siNA duplex. The specific modifications shown in thefigure can be utilized alone or in combination with other modificationsof the figure, in addition to other modifications and features describedherein with reference to any siNA molecule of the invention. In thefigure, N stands for any nucleotide or optionally a non-nucleotide asdescribed here. The upper strand, having B—N_(X3)—(N)_(X2)—B-3′ is thesense (or passenger) strand of the siNA, whereas the lower strand,having B(N)_(X1)—N_(X4)—[N]_(X5)-5′ is the antisense (or guide) strandof the siNA. Nucleotides (or optional non-nucleotides) of internalportions of the sense strand are designated N_(X3) and nucleotides (oroptional non-nucleotides) of internal portions of the antisense strandare designated N_(X4). Nucleotides (or optional non-nucleotides) of theinternal portions are generally base paired between the two strands, butcan optionally lack base pairing (e.g. have mismatches or gaps) in someembodiments. Nucleotides (or optional non-nucleotides) of overhangregions are designated by parenthesis (N). Nucleotides of the5′-terminal portion of the antisense strand are designated [N]. Terminalcaps are optionally present at the 5′ and/or 3′ end of the sense strandand further optionally present at the 3′-end of the antisense strand.Generally, each strand can independently range from about 15 to about 30nucleotides in length, but can vary depending on the presence of anyoverhang nucleotides. In certain embodiments, X1 and X2 areindependently integers from 0 to 4; X3 is an integer from 15 to 30; X4is an integer from 9 to 30; X5 is an integer from 0 to 6, provided thatthe sum of X4 and X5 is 15-30. Various modifications are shown for thenucleotides of the sense and antisense strands of the siNA constructs.The (N) overhang nucleotide positions can be chemically modified asdescribed herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy, LNA,universal bases etc.) and can be either derived from a correspondingtarget nucleic acid sequence or not. The constructs shown in the figurecan also comprise phosphorothioate linkages as described herein. Forexample, phosphorothioate linkages can exist between any N, (N), and/or[N] positions. Such phosphorothioate incorporation can be utilizedbetween purine “R” and pyrimidine “Y” positions, or for stabilization ofpyrimidine linkages in general. Furthermore, although not depicted onthe Figure, the constructs shown in the figure can optionally include aribonucleotide at the 9th position from the 5′-end of the sense strandor the 11^(th) position based on the 5′-end of the guide strand bycounting 11 nucleotide positions in from the 5′-terminus of the guidestrand. Similarly, the antisense strand can include a ribonucleotide atthe 14^(th) position from the 5′-end, or alternately can be selected ordesigned so that a 2′-O-alkyl nucleotide (e.g., a 2′-O-methyl purine) isnot present at this position. Furthermore, although not shown in theFigure, the 5′-terminal position of the antisense strand can comprise aterminal phosphate group as described herein. The antisense strandgenerally comprises sequence complementary to any target nucleic acidsequence of the invention, such as those set forth in Table 1a herein.

FIG. 3 shows non-limiting examples of certain combinations ofmodifications applied to the representative siNA duplex described inFIG. 2. The table shown below the representative structure providesspecific combinations of (N)_(X1), (N)_(X2), N_(X3), N_(X4), and/or[N]_(X5) nucleotide (and optional non-nucleotide) positions. Forexample, combinations of 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more)N_(X3) and 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) N_(X4)pyrimidine “Y” and purine “R” nucleotides are specified, each of whichcan independently have specific (N)_(X1), and/or (N)_(X2), substitutionsas shown in the figure, in addition to optional phosphorothioatesubstitutions. The 5′-terminal antisense strand [N] nucleotides aregenerally ribonucleotides, but can also be modified or unmodifieddepending on if they are purine “R” or pyrimidine “Y” nucleotides

FIG. 4A-C shows non-limiting examples of different siNA constructs ofthe invention. The criteria of the representative structures shown inFIGS. 2 and 3 can be applied to any of the structures shown in FIG.4A-C.

The examples shown in FIG. 4A (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

The examples shown in FIG. 4B represent different variations ofdouble-stranded nucleic acid molecule of the invention, such asmicroRNA, that can include overhangs, bulges, loops, and stem-loopsresulting from partial complementarity. Such motifs having bulges,loops, and stem-loops are generally characteristics of miRNA. Thebulges, loops, and stem-loops can result from any degree of partialcomplementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more nucleotides in one or both strands of thedouble-stranded nucleic acid molecule of the invention.

The example shown in FIG. 4C represents a model double-stranded nucleicacid molecule of the invention comprising a 19 base pair duplex of two21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand(1) represents the sense strand (passenger strand), the middle strand(2) represents the antisense (guide strand), and the lower strand (3)represents a target polynucleotide sequence. The dinucleotide overhangs(NN) can comprise a sequence derived from the target polynucleotide. Forexample, the 3′-(NN) sequence in the guide strand can be complementaryto the 5′-[NN] sequence of the target polynucleotide. In addition, the5′-(NN) sequence of the passenger strand can comprise the same sequenceas the 5′-[NN] sequence of the target polynucleotide sequence. In otherembodiments, the overhangs (NN) are not derived from the targetpolynucleotide sequence, for example where the 3′-(NN) sequence in theguide strand are not complementary to the 5′-[NN] sequence of the targetpolynucleotide and the 5′-(NN) sequence of the passenger strand cancomprise different sequence from the 5′-[NN] sequence of the targetpolynucleotide sequence. In additional embodiments, any (NN) nucleotidesare chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or other modifications herein. Furthermore, the passenger strand cancomprise a ribonucleotide position N of the passenger strand. For therepresentative 19 base pair 21 mer duplex shown, position N can be 9nucleotides in from the 3′ end of the passenger strand. However, induplexes of differing length, the position N is determined based on the5′-end of the guide strand by counting 11 nucleotide positions in fromthe 5′-terminus of the guide strand and picking the corresponding basepaired nucleotide in the passenger strand. Cleavage by Ago2 takes placebetween positions 10 and 11 as indicated by the arrow. In additionalembodiments, there are two ribonucleotides, NN, at positions 10 and 11based on the 5′-end of the guide strand by counting 10 and 11 nucleotidepositions in from the 5′-terminus of the guide strand and picking thecorresponding base paired nucleotides in the passenger strand.

FIG. 5 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the 5′and/or 3′-ends of siNA sequences of the invention, including (1)[3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide (whenX═O). In addition to modified and unmodified backbone chemistriesindicated in the figure, these chemistries can be combined withdifferent sugar and base nucleotide modifications as described herein.

FIG. 6 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistant while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct is tested in an appropriate system (e.g., humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay and/or against endogenous mRNA). Lead siNA constructs arethen identified which possess a particular characteristic whilemaintaining RNAi activity, and can be further modified and assayed onceagain. This same approach can be used to identify siNA-conjugatemolecules with improved pharmacokinetic profiles, delivery, and RNAiactivity.

FIG. 7 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 8 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 9 shows a non-limiting example of a cholesterol linkedphosphoramidite that can be used to synthesize cholesterol conjugatedsiNA molecules of the invention. An example is shown with thecholesterol moiety linked to the 5′-end of the sense strand of an siNAmolecule.

FIG. 10 depicts an embodiment of 5′ and 3′ inverted abasic cap linked toa nucleic acid strand.

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

The following terminology and definitions apply as used in the presentapplication.

The term “abasic” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to sugar moieties lackinga nucleobase or having a hydrogen atom (H) or other non-nucleobasechemical groups in place of a nucleobase at the 1′ position of the sugarmoiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In oneembodiment, an abasic moiety of the invention is a ribose, deoxyribose,or dideoxyribose sugar.

The term “acyclic nucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to anynucleotide having an acyclic ribose sugar, for example where any of theribose carbon/carbon or carbon/oxygen bonds are independently or incombination absent from the nucleotide.

The term “alkyl” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a saturated orunsaturated hydrocarbons, including straight-chain, branched-chain,alkenyl, alkynyl groups and cyclic groups, but excludes aromatic groups.Notwithstanding the foregoing, alkyl also refers to non-aromaticheterocyclic groups. Preferably, the alkyl group has 1 to 12 carbons.More preferably, it is a lower alkyl of from 1 to 7 carbons, morepreferably 1 to 4 carbons. The alkyl group can be substituted orunsubstituted. When substituted, the substituted group(s) is preferably,hydroxyl, halogen, cyano, C1-C4 alkoxy, ═O, ═S, NO₂, SH, NH₂, or NR₁R₂,where R₁ and R₂ independently are H or C1-C4 alkyl.

The phrase “agents that interfere with cell cycle checkpoints” refers tocompounds that inhibit protein kinases that transduce cell cyclecheckpoint signals, thereby sensitizing the cancer cell to DNA damagingagents.

The phrase “agents that interfere with receptor tyrosine kinases (RTKs)”refers to compounds that inhibit RTKs and therefore inhibit mechanismsinvolved in oncogenesis and tumor progression.

The phrase “androgen receptor modulators” refers to compounds thatinterfere or inhibit the binding of androgens to the receptor,regardless of mechanism.

The phrase “angiogenesis inhibitors” refers to compounds that inhibitthe formation of new blood vessels, regardless of mechanism.

The term “aryl” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to an aromatic group thathas at least one ring having a conjugated pi electron system andincludes carbocyclic aryl, heterocyclic aryl and biaryl groups, all ofwhich can be optionally substituted. The preferred substituent(s) ofaryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, C1-C4alkoxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, NH₂, and NR₁R₂groups, where R₁ and R₂ independently are H or C1-C4 alkyl.

The term “alkylaryl” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to an alkylgroup (as described above) covalently joined to an aryl group (asdescribed above). Carbocyclic aryl groups are groups wherein the ringatoms on the aromatic ring are all carbon atoms. The carbon atoms areoptionally substituted. Heterocyclic aryl groups are groups having from1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainderof the ring atoms are carbon atoms. Suitable heteroatoms include oxygen,sulfur, and nitrogen, and examples of heterocyclic aryl groups havingsuch heteroatoms include furanyl, thienyl, pyridyl, pyrrolyl, N-loweralkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, alloptionally substituted. Preferably, the alkyl group is a C1-C4 alkylgroup.

The term “amide” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to an —C(O)—NH—R, where Ris either alkyl, aryl, alkylaryl or hydrogen.

The phrase “antisense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to a nucleotide sequence ofan siNA molecule having complementarity to a target nucleic acidsequence. In addition, the antisense region of an siNA molecule canoptionally comprise a nucleic acid sequence having complementarity to asense region of the siNA molecule. In one embodiment, the antisenseregion of the siNA molecule is referred to as the antisense strand orguide strand.

The phrase “asymmetric hairpin” refers to a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

The term “biodegradable” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to degradationin a biological system, for example, enzymatic degradation or chemicaldegradation.

The term “biodegradable linker” as used herein refers to its meaning asis generally accepted in the art. With reference to exemplary nucleicacid molecules of the invention, the term refers to a linker moleculethat is designed to connect one molecule to another molecule, and whichis susceptible to degradation in a biological system. The linker can bea nucleic acid or non-nucleic acid based linker. For example, abiodegradable linker can be used to attach a ligand or biologicallyactive molecule to an siNA molecule of the invention. Alternately, abiodegradable linker can be used to connect the sense and antisensestrands of an siNA molecule of the invention. The biodegradable linkeris designed such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bemodulated by using various chemistries, for example combinations ofribonucleotides, deoxyribonucleotides, and chemically modifiednucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. The biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example, anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus-based linkage, for example, aphosphoramidate or phosphodiester linkage. The biodegradable nucleicacid linker molecule can also comprise nucleic acid backbone, nucleicacid sugar, or nucleic acid base modifications.

The phrase “biologically active molecule” as used herein refers to itsmeaning as is generally accepted in the art. With reference to exemplarynucleic acid molecules of the invention, the term refers to compounds ormolecules that are capable of eliciting or modifying a biologicalresponse in a system and/or are capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules. Examples of biologically active molecules, include siNAmolecules alone or in combination with other molecules including, butnot limited to therapeutically active molecules such as antibodies,cholesterol, hormones, antivirals, peptides, proteins,chemotherapeutics, small molecules, vitamins, co-factors, nucleosides,nucleotides, oligonucleotides, enzymatic nucleic acids, antisensenucleic acids, triplex forming oligonucleotides, polyamines, polyamides,polyethylene glycol, other polyethers, 2-5A chimeras, siNA, dsRNA,allozymes, aptamers, decoys and analogs thereof.

The phrase “biological system” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to material,in a purified or unpurified form, from biological sources including, butnot limited to, human or animal, wherein the system comprises thecomponents required for RNAi activity. Thus, the phrase includes, forexample, a cell, tissue, subject, or organism, or extract thereof. Theterm also includes reconstituted material from a biological source.

The phrase “blunt end” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to termini of adouble-stranded siNA molecule having no overhanging nucleotides. Forexample, the two strands of a double-stranded siNA molecule having bluntends align with each other with matched base-pairs without overhangingnucleotides at the termini. A siNA duplex molecule of the invention cancomprise blunt ends at one or both termini of the duplex, such astermini located at the 5′-end of the antisense strand, the 5′-end of thesense strand, or both termini of the duplex.

The term “cap” also referred to herein as “terminal cap,” as used hereinrefers to its meaning as is generally accepted in the art. Withreference to exemplary nucleic acid molecules of the invention, the termrefers to a moiety, which can be a chemically modified nucleotide ornon-nucleotide that can be incorporated at one or more termini of one ormore nucleic acid molecules of the invention. These terminalmodifications protect the nucleic acid molecule from exonucleasedegradation, and can help in delivery and/or localization within a cell.The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminal(3′-cap) or can be present on both termini of any nucleic acid moleculeof the invention. A cap can be present at the 5′-end, 3-end and/or 5′and 3′-ends of the sense strand of a nucleic acid molecule of theinvention. Additionally, a cap can optionally be present at the 3′-endof the antisense strand of a nucleic acid molecule of the invention. Innon-limiting examples, the 5′-cap includes, but is not limited to, LNA;glyceryl; inverted deoxy abasic residue (moiety); 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide;carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide;3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety. Non-limiting examplesof the 3′-cap include, but are not limited to, LNA; glyceryl; inverteddeoxy abasic residue (moiety); 4′, 5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide; carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate;3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate;phosphorothioate and/or phosphorodithioate; bridging or non bridgingmethylphosphonate; and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein). FIG. 5 shows some non-limiting examples of various caps.

The term “cell” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary nucleic acid moleculesof the invention, the term is used in its usual biological sense, anddoes not refer to an entire multicellular organism, e.g., specificallydoes not refer to a human being. The cell can be present in an organism,e.g., birds, plants and mammals, such as humans, cows, sheep, apes,monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cellcan be of somatic or germ line origin, totipotent or pluripotent,dividing or non-dividing. The cell can also be derived from or cancomprise a gamete or embryo, a stem cell, or a fully differentiatedcell.

The phrase “chemical modification” as used herein refers to its meaningas is generally accepted in the art. With reference to exemplary nucleicacid molecules of the invention, the term refers to any modification ofthe chemical structure of the nucleotides that differs from nucleotidesof native siRNA or RNA in general. The term “chemical modification”encompasses the addition, substitution, or modification of native siRNAor RNA at the sugar, base, or internucleotide linkage, as describedherein or as is otherwise known in the art. In certain embodiments, theterm “chemical modification” can refer to certain forms of RNA that arenaturally occurring in certain biological systems, for example2′-O-methyl modifications or inosine modifications.

The term “complementarity” or “complementary” as used herein refers toits meaning as is generally accepted in the art. With reference toexemplary nucleic acid molecules of the invention, the terms generallyrefer to the formation or existence of hydrogen bond(s) between onenucleic acid sequence and another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of bonding asdescribed herein. In reference to the nucleic molecules of the presentinvention, the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Perfectcomplementary means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. Partial complementarity can includevarious mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non-basedpaired nucleotides) within the nucleic acid molecule, which can resultin bulges, loops, or overhangs that result between the sense strand orsense region and the antisense strand or antisense region of the nucleicacid molecule or between the antisense strand or antisense region of thenucleic acid molecule and a corresponding target nucleic acid molecule.Such partial complementarity can be represented by a % complementaritythat is determined by the number of non-base paired nucleotides, i.e.,about 50%, 60%, 70%, 80%, 90% etc. depending on the total number ofnucleotides involved. Such partial complementarity is permitted to theextent that the nucleic acid molecule (e.g., siNA) maintains itsfunction, for example the ability to mediate sequence specific RNAi.

The terms “composition” or “formulation” as used herein refer to theirgenerally accepted meaning in the art. These terms generally refer to acomposition or formulation, such as in a pharmaceutically acceptablecarrier or diluent, in a form suitable for administration, e.g.,systemic or local administration, into a cell or subject, including, forexample, a human. Suitable forms, in part, depend upon the use or theroute of entry, for example oral, transdermal, inhalation, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell (i.e., a cell to which the negativelycharged nucleic acid is desirable for delivery). For example,compositions injected into the blood stream should be soluble. Otherfactors are known in the art, and include considerations such astoxicity and forms that prevent the composition or formulation fromexerting its effect. As used herein, pharmaceutical formulations includeformulations for human and veterinary use. Non-limiting examples ofagents suitable for formulation with the nucleic acid molecules of theinstant invention include: Lipid Nanoparticles (see for example Sempleet al., 2010, Nat Biotechnol., February; 28(2):172-6.); P-glycoproteininhibitors (such as Pluronic P85); biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release delivery(Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loadednanoparticles, such as those made of polybutylcyanoacrylate. Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058. A “pharmaceutically acceptable composition” or“pharmaceutically acceptable formulation” can refer to a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention to the physical location mostsuitable for their desired activity.

The phrase “cytotoxic/cytostatic agents” refer to compounds that causecell death or inhibit cell proliferation primarily by interferingdirectly with the cell's functioning or inhibit or interfere with cellmytosis, including alkylating agents, tumor necrosis factors,intercalators, hypoxia activatable compounds, microtubuleinhibitors/microtubule-stabilizing agents, inhibitors of mitotickinesins, inhibitors of histone deacetylase, inhibitors of kinasesinvolved in mitotic progression, antimetabolites; biological responsemodifiers; hormonal/anti-hormonal therapeutic agents, hematopoieticgrowth factors, monoclonal antibody targeted therapeutic agents,topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligaseinhibitors.

The phrase “estrogen receptor modulators” refers to compounds thatinterfere with or inhibit the binding of estrogen to the receptor,regardless of mechanism.

The term “gene” or “target gene” as used herein refers to their meaningas is generally accepted in the art. The terms generally refer a nucleicacid (e.g., DNA or RNA) sequence that comprises partial length or entirelength coding sequences necessary for the production of a polypeptide.The target gene can also include the UTR or non-coding region of thenucleic acid sequence. A gene or target gene can also encode afunctional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporalRNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), shortinterfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA(rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-codingRNAs can serve as target nucleic acid molecules for siNA mediated RNAinterference in modulating the activity of fRNA or ncRNA involved infunctional or regulatory cellular processes. Aberrant fRNA or ncRNAactivity leading to disease can therefore be modulated by siNA moleculesof the invention. siNA molecules targeting fRNA and ncRNA can also beused to manipulate or alter the genotype or phenotype of a subject,organism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts. For a review, see for example Snyderand Gerstein, 2003, Science, 300, 258-260.

The phrase “HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. The term HMG-CoA reductaseinhibitor as used herein includes all pharmaceutically acceptablelactone and open-acid forms (i.e., where the lactone ring is opened toform the free acid) as well as salt and ester forms of compounds thathave HMG-CoA reductase inhibitory activity, and therefore the use ofsuch salts, esters, open-acid and lactone forms is included within thescope of this invention.

The term “HBV” refers to Hepatitis B Virus, which is gene that encodesHBV proteins, HBV peptides, HBV polypeptides, HBV regulatorypolynucleotides (e.g., HBV miRNAs and siNAs), mutant HBV genes, andsplice variants of a HBV genes, as well as other genes involved in HBVpathways of gene expression and/or activity. Thus, each of theembodiments described herein with reference to the term “HBV” areapplicable to all of the protein, peptide, polypeptide, and/orpolynucleotide molecules covered by the term “HBV”, as that term isdefined herein. Comprehensively, such gene targets are also referred toherein generally as “target” sequences (including the target sequenceslisted in Table 1a).

The phrase “highly conserved sequence region” refers to a nucleotidesequence of one or more regions in a target gene that does not varysignificantly from one generation to the other or from one biologicalsystem to the other.

The phrase “homologous sequence” as used herein refers to its meaning asis generally accepted in the art. The term generally refers a nucleotidesequence that is shared by one or more polynucleotide sequences, such asgenes, gene transcripts and/or non-coding polynucleotides. For example,a homologous sequence can be a nucleotide sequence that is shared by twoor more genes encoding related but different proteins, such as differentmembers of a gene family, different protein epitopes, different proteinisoforms or completely divergent genes. A homologous sequence can be anucleotide sequence that is shared by two or more non-codingpolynucleotides, such as noncoding DNA or RNA, regulatory sequences,introns, and sites of transcriptional control or regulation. Homologoussequences can also include sequence regions shared by more than onepolynucleotide sequence. Homology does not need to be perfect identity(100%), as partially homologous sequences are also contemplated by andwithin the scope of the instant invention (e.g., at least 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).Percent homology is the number of matching nucleotides between twosequences divided by the total length being compared, multiplied by 100.

The phrase “improved RNAi activity” refers to an increase in RNAiactivity measured in vitro and/or in vivo, where the RNAi activity is areflection of both the ability of the siNA to mediate RNAi and thestability of the siNAs of the invention. In this invention, the productof these activities can be increased in vitro and/or in vivo compared toan all RNA siNA or an siNA containing a plurality of ribonucleotides. Insome cases, the activity or stability of the siNA molecule can bedecreased (i.e., less than ten-fold), but the overall activity of thesiNA molecule is enhanced in vitro and/or in vivo.

The terms “inhibit,” “down-regulate,” or “reduce” as used herein refersto its meaning as is generally accepted in the art. With reference toexemplary nucleic acid molecules of the invention, he term generallyrefers the reduction in the expression of the gene, or level of RNAmolecules or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits, below that observed in the absence of the nucleic acidmolecules (e.g., siNA) of the invention. Down-regulation can also beassociated with post-transcriptional silencing, such as, RNAi mediatedcleavage or by alteration in DNA methylation patterns or DNA chromatinstructure. Inhibition, down-regulation or reduction with an siNAmolecule can be in reference to an inactive molecule, an attenuatedmolecule, an siNA molecule with a scrambled sequence, or an siNAmolecule with mismatches or alternatively, it can be in reference to thesystem in the absence of the nucleic acid.

The phrase “inhibitors of cell proliferation and survival signalingpathway” refers to pharmaceutical agents that inhibit cell surfacereceptors and signal transduction cascades downstream of those surfacereceptors.

The term “integrin blockers” refers to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe α _(ω) β₃ integrin, to compounds which selectively antagonize,inhibit or counteract binding of a physiological ligand to the α _(ω) β₅integrin, to compounds which antagonize, inhibit or counteract bindingof a physiological ligand to both the α _(ω) β₃ integrin and the α _(ω)β₅ integrin, and to compounds which antagonize, inhibit or counteractthe activity of the particular integrin(s) expressed on capillaryendothelial cells. The term also refers to antagonists of the α _(ω) β₆,α _(ω) β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The term alsorefers to antagonists of any combination of α _(ω) β₃, α _(ω) β₅, α _(ω)β₆, α _(ω) β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

The terms “intermittent” or “intermittently” as used herein refers toits meaning as is generally accepted in the art. The term generallyrefers to periodic stopping and starting at either regular or irregularintervals.

The terms “internucleoside linkage” or “internucleoside linker” or“internucleotide linkage” or “internucleotide linker” are used hereininterchangeably and refer to any linker or linkage between twonucleoside units, as is known in the art, including, for example, butnot limitation, phosphate, analogs of phosphate, phosphonate, guanidium,hydroxylamine, hydroxylhydrazinyl, amide, carbamate, alkyl, andsubstituted alkyl linkages. The internucleoside linkages constitute thebackbone of a nucleic acid molecule.

The terms “mammalian” or “mammal” as used herein refers to its meaningas is generally accepted in the art. The term generally refers to anywarm blooded vertebrate species, such as a human, mouse, rat, dog, cat,hamster, guinea pig, rabbit, livestock, and the like.

The phrase “metered dose inhaler” or MDI refers to a unit comprising acan, a secured cap covering the can and a formulation metering valvesituated in the cap. MDI systems includes a suitable channeling device.Suitable channeling devices comprise for example, a valve actuator and acylindrical or cone-like passage through which medicament can bedelivered from the filled canister via the metering valve to the nose ormouth of a patient such as a mouthpiece actuator.

The term “microRNA” or “miRNA” as used herein refers to its meaning asis generally accepted in the art. The term generally refers a smalldouble-stranded RNA that regulates the expression of target messengerRNAs either by mRNA cleavage, translational repression/inhibition orheterochromatic silencing (see for example Ambros, 2004, Nature, 431,350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, VirusResearch., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; Yinget al., 2004, Gene, 342, 25-28; and Sethupathy et al., 2006, RNA,12:192-197).

The term “modulate” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary nucleic acid moleculesof the invention, the term refers to when the expression of a gene, orlevel of one or more RNA molecules (coding or non-coding), or activityof one or more RNA molecules or proteins or protein subunits, isup-regulated or down-regulated, such that expression, level, or activityis greater than or less than that observed in the absence of themolecule that effects modulation. For example, the term “modulate” insome embodiments can refer to inhibition and in other embodiments canrefer to potentiation or up-regulation, e.g., of gene expression.

The phrase “modified nucleotide” as used herein refers to its meaning asis generally accepted in the art. The term generally refers anucleotide, which contains a modification in the chemical structure ofthe base, sugar and/or phosphate of the unmodified (or natural)nucleotide as is generally known in the art. Non-limiting examples ofmodified nucleotides are described herein and in U.S. application Ser.No. 12/064,014.

The phrase “NSAIDs that are selective COX-2 inhibitors” for purposesherein, refers to NSAIDs, which possess a specificity for inhibitingCOX-2 over COX-1 of at least 100 fold as measured by the ratio of IC₅₀for COX-2 over IC₅₀ for COX-1 evaluated by cell or microsomal assays.

The phrase “non-base paired” refers to nucleotides that are not basepaired between the sense strand or sense region and the antisense strandor antisense region of an double-stranded siNA molecule; and can includefor example, but not limitation, mismatches, overhangs, single strandedloops, etc.

The term “non-nucleotide” refers to any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, such as for example but not limitation abasic moietiesor alkyl chains. The group or compound is “abasic” in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine and therefore lacks a nucleobase atthe 1′-position.

The term “nucleotide” is used as is generally recognized in the art.Nucleotides generally comprise a nucleobase, a sugar, and aninternucleoside linkage, e.g., a phosphate. The base can be a naturalbases (standard), modified bases, or a base analog, as are well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Additionally, the nucleotides can be unmodifiedor modified at the sugar, internucleoside linkage, and/or base moiety,(also referred to interchangeably as nucleotide analogs, modifiednucleotides, non-natural nucleotides, non-standard nucleotides andothers; see, for example, U.S. application Ser. No. 12/064,014.

The term “overhang” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary double stranded nucleicacid molecules, the term generally refers to the terminal portion of anucleotide sequence that is not base paired between the two strands of adouble-stranded nucleic acid molecule (see for example, FIG. 4).Overhangs, when present, are typically at the 3′-end of one or bothstrands in a siNA duplex.

The term “parenteral” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers methods ortechniques of administering a molecule, drug, agent, or compound in amanner other than through the digestive tract, and includesepicutaneous, subcutaneous, intravascular (e.g., intravenous),intramuscular, or intrathecal injection or infusion techniques and thelike.

The phrase “pathway target” refers to any target involved in pathways ofgene expression or activity. For example, any given target can haverelated pathway targets that can include upstream, downstream, ormodifier genes in a biologic pathway. These pathway target genes canprovide additive or synergistic effects in the treatment of diseases,conditions, and traits herein.

The term “phosphorothioate” refers to an internucleotide phosphatelinkage comprising one or more sulfur atoms in place of an oxygen atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

“Prenyl-protein transferase inhibitor” refers to a compound thatinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase).

The phrase “retinoid receptor modulators” refers to compounds thatinterfere or inhibit the binding of retinoids to the receptor,regardless of mechanism.

The term “ribonucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.

The term “RNA” as used herein refers to its generally accepted meaningin the art. Generally, the term RNA refers to a molecule comprising atleast one ribofuranoside moiety. The term can include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas altered RNA that differs from naturally occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siNA or internally, for exampleat one or more nucleotides of the RNA. Nucleotides in the RNA moleculesof the instant invention can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

The phrase “RNA interference” or term “RNAi” refer to the biologicalprocess of inhibiting or down regulating gene expression in a cell, asis generally known in the art, and which is mediated by shortinterfering nucleic acid molecules, see for example Zamore and Haley,2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science,309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001,Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; andKreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).Additionally, the term RNAi is meant to be equivalent to other termsused to describe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, siNA moleculesof the invention can be used to epigenetically silence genes at eitherthe post-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation patterns to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237). In another non-limiting example, modulation of geneexpression by siNA molecules of the invention can result from siNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, orvia translational inhibition, as is known in the art or modulation canresult from transcriptional inhibition (see for example Janowski et al.,2005, Nature Chemical Biology, 1, 216-222).

The phrase “RNAi inhibitor” refers to any molecule that can downregulate, reduce or inhibit RNA interference function or activity in acell or organism. An RNAi inhibitor can down regulate, reduce or inhibitRNAi (e.g., RNAi mediated cleavage of a target polynucleotide,translational inhibition, or transcriptional silencing) by interactionwith or interfering with the function of any component of the RNAipathway, including protein components such as RISC, or nucleic acidcomponents such as miRNAs or siRNAs. A RNAi inhibitor can be an siNAmolecule, an antisense molecule, an aptamer, or a small molecule thatinteracts with or interferes with the function of RISC, a miRNA, or ansiRNA or any other component of the RNAi pathway in a cell or organism.By inhibiting RNAi (e.g., RNAi mediated cleavage of a targetpolynucleotide, translational inhibition, or transcriptional silencing),a RNAi inhibitor of the invention can be used to modulate (e.g.,up-regulate or down regulate) the expression of a target gene.

The phrase “sense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to a nucleotide sequence ofan siNA molecule having complementarity to an antisense region of thesiNA molecule. In addition, the sense region of an siNA molecule cancomprise a nucleic acid sequence having homology or sequence identitywith a target nucleic acid sequence. In one embodiment, the sense regionof the siNA molecule is also referred to as the sense strand orpassenger strand.

The phrases “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” refer to any nucleic acid moleculecapable of inhibiting or down regulating gene expression or viralreplication by mediating RNA interference (“RNAi”) or gene silencing ina sequence-specific manner. These terms can refer to both individualnucleic acid molecules, a plurality of such nucleic acid molecules, orpools of such nucleic acid molecules. The siNA can be a double-strandednucleic acid molecule comprising self-complementary sense and antisensestrands, wherein the antisense strand comprises a nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises anucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be a polynucleotide with a duplex,asymmetric duplex, hairpin or asymmetric hairpin secondary structure,having self-complementary sense and antisense regions, wherein theantisense region comprises a nucleotide sequence that is complementaryto a nucleotide sequence in a separate target nucleic acid molecule or aportion thereof and the sense region comprises a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be a circular single-stranded polynucleotide having two ormore loop structures and a stem comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense regioncomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof, and wherein the circular polynucleotidecan be processed either in vivo or in vitro to generate an active siNAmolecule capable of mediating RNAi. The siNA can also comprise asingle-stranded polynucleotide having a nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single-stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for example,Martinez et al., 2002, Cell, 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.

The term “subject” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers an organism to which thenucleic acid molecules of the invention can be administered. A subjectcan be a mammal or mammalian cells, including a human or human cells.The term also refers to an organism, which is a donor or recipient ofexplanted cells or the cells themselves.

The phrase “systemic administration” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersin vivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body.

The term “target” as it refers to HBV refers to any HBV target protein,peptide, or polypeptide, such as encoded by Genbank Accession Nos. shownin Table 7. The term also refers to nucleic acid sequences or targetpolynucleotide sequence encoding any target protein, peptide, orpolypeptide, such as proteins, peptides, or polypeptides encoded bysequences having Genbank Accession Nos. shown in Table 7. The target ofinterest can include target polynucleotide sequences, such as target DNAor target RNA. The term “target” is also meant to include othersequences, such as differing isoforms, mutant target genes, splicevariants of target polynucleotides, target polymorphisms, and non-coding(e.g., ncRNA, miRNA, stRNA, sRNA) or other regulatory polynucleotidesequences as described herein.

The phrase “target site” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a sequencewithin a target nucleic acid molecule, (e.g., RNA) that is “targeted”,e.g., for cleavage mediated by an siNA construct, which containssequences within its antisense region that are complementary to thetarget sequence.

The phrase “therapeutically effective amount” as used herein refers toits meaning as is generally accepted in the art. The term generallyrefers to the amount of the compound or composition that will elicit thebiological or medical response of a cell, tissue, system, animal orhuman that is be sought by the researcher, veterinarian, medical doctoror other clinician. For example, if a given clinical treatment isconsidered effective when there is at least a 25% reduction in ameasurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is that amount necessary to effect at least a 25%reduction in that parameter.

The phrase “universal base” as used herein refers to its meaning as isgenerally accepted in the art. The term universal base generally refersto nucleotide base analogs that form base pairs with each of the naturalDNA/RNA bases with little or no discrimination between them.Non-limiting examples of universal bases include C-phenyl, C-naphthyland other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole as known in the art (see for example,Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “up-regulate” as used herein refers to its meaning as isgenerally accepted in the art. With reference to exemplary nucleic acidmolecules of the invention, the term refers to an increase in theexpression of a gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more RNAs, proteins or protein subunits, above that observedin the absence of the nucleic acid molecules (e.g., siNA) of theinvention. In certain instances, up-regulation or promotion of geneexpression with an siNA molecule is above that level observed in thepresence of an inactive or attenuated molecule. In other instances,up-regulation or promotion of gene expression with siNA molecules isabove that level observed in the presence of, for example, an siNAmolecule with scrambled sequence or with mismatches. In still otherinstances, up-regulation or promotion of gene expression with a nucleicacid molecule of the instant invention is greater in the presence of thenucleic acid molecule than in its absence. In some instances,up-regulation or promotion of gene expression is associated withinhibition of RNA mediated gene silencing, such as RNAi mediatedcleavage or silencing of a coding or non-coding RNA target that downregulates, inhibits, or silences the expression of the gene of interestto be up-regulated. The down regulation of gene expression can, forexample, be induced by a coding RNA or its encoded protein, such asthrough negative feedback or antagonistic effects. The down regulationof gene expression can, for example, be induced by a non-coding RNAhaving regulatory control over a gene of interest, for example bysilencing expression of the gene via translational inhibition, chromatinstructure, methylation, RISC mediated RNA cleavage, or translationalinhibition. As such, inhibition or down regulation of targets that downregulate, suppress, or silence a gene of interest can be used toup-regulate expression of the gene of interest toward therapeutic use.

The term “vector” as used herein refers to its meaning as is generallyaccepted in the art. The term vector generally refers to any nucleicacid- and/or viral-based expression system or technique used to deliverone or more nucleic acid molecules.

B. siNA Molecules of the Invention

The present invention provides compositions and methods comprising siNAstargeted to HBV that can be used to treat diseases, e.g., liver diseaseor malignancies and/or cancers associated with HBV expression or otherconditions that result from hepatitis B infection. In particular aspectsand embodiments of the invention, the nucleic acid molecules of theinvention comprise at least a 15 nucleotide sequence of the sequencesshown in Table 1a and Table 1b. The siNAs can be provided in severalforms. For example, the siNA can be isolated as one or more siNAcompounds, or it may be in the form of a transcriptional cassette in aDNA plasmid. The siNA may also be chemically synthesized and can includemodifications as shown, for example, but not limitation, in Table 1c andTable 8. Thus, in various embodiments, at least one strand or region ofthe nucleic acids of the invention comprises at least a 15 nucleotidesequence selected from the group of sequences consisting of SEQ IDNOS:1-502. The siNAs can be administered alone or co-administered withother siNA molecules or with conventional agents that treat a HBVrelated disease or condition.

The siNA molecules of the invention can be used to mediate genesilencing, specifically HBV, via interaction with RNA transcripts oralternately by interaction with particular gene sequences, wherein suchinteraction results in modulation of gene silencing either at thetranscriptional level or post-transcriptional level such as, forexample, but not limited to, RNAi or through cellular processes thatmodulate the chromatin structure or methylation patterns of the targetand prevent transcription of the target gene, with the nucleotidesequence of the target thereby mediating silencing. More specifically,the target is any of HBV RNA, DNA, or mRNA,

In one aspect, the invention provides short interfering nucleic acid(siNA) molecules for inhibiting the expression of the HBV gene in a cellor mammal. The siNA can be single-stranded or double-stranded. Whendouble-stranded, the siNA comprising a sense and an antisense stand. Theantisense strand is complementary to at least a part of an mRNA formedin the expression of the HBV gene. The sense strand comprises a regionthat is complementary to the antisense strand. In specific embodiments,the antisense strand comprises at least a 15 nucleotide sequence of anantisense sequence listed in Table 1b. Generally, the double-strandedsiNA comprises at least a 15 nucleotide sequence of the sense strand inTable 1b and at least a 15 nucleotide sequence of the antisense strandin Table 1b. One or more of the nucleotides of the siNAs of theinvention are optionally modified. In further embodiments havingmodifications, some siNAs of the invention comprises at least onenucleotide sequence selected from the groups of sequences provide inTable 1c. In other embodiments, the siNA comprises at least twosequences selected from the group of sequences provided in Table 1c,wherein one of the at least two sequences is complementary to another ofthe at least two sequences and one of the at least two sequences iscomplementary to a sequence of a mRNA generated in the expression of theHBV gene. Examples of certain modified siNAs of the invention are inTable 1c.

The double stranded RNA molecules of the invention can comprise twodistinct and separate strands that can be symmetric or asymmetric andare complementary, i.e., two single-stranded RNA molecules, or cancomprise one single-stranded molecule in which two complementaryportions, e.g., a sense region and an antisense region, are base-paired,and are covalently linked by one or more single-stranded “hairpin” areas(i.e. loops) resulting in, for example, a single-stranded short-hairpinpolynucleotide or a circular single-stranded polynucleotide.

The linker can be polynucleotide linker or a non-nucleotide linker. Insome embodiments, the linker is a non-nucleotide linker. In someembodiments, a hairpin or circular siNA molecule of the inventioncontains one or more loop motifs, wherein at least one of the loopportions of the siNA molecule is biodegradable. For example, asingle-stranded hairpin siNA molecule of the invention is designed suchthat degradation of the loop portion of the siNA molecule in vivo cangenerate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising 1, 2, 3 or 4nucleotides. Or alternatively, a circular siNA molecule of the inventionis designed such that degradation of the loop portions of the siNAmolecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In symmetric siNA molecules of the invention, each strand, the sense(passenger) strand and antisense (guide) strand, are independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length. Generally, each strand ofthe symmetric siNA molecules of the invention are about 19-24 (e.g.,about 19, 20, 21, 22, 23 or 24) nucleotides in length.

In asymmetric siNA molecules, the antisense region or strand of themolecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length,wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides in length. Generally, each strand of the asymmetric siNAmolecules of the invention is about 19-24 (e.g., about 19, 20, 21, 22,23 or 24) nucleotides in length.

In yet other embodiments, siNA molecules of the invention comprisesingle stranded hairpin siNA molecules, wherein the siNA molecules areabout 25 to about 70 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length.

In still other embodiments, siNA molecules of the invention comprisesingle-stranded circular siNA molecules, wherein the siNA molecules areabout 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70)nucleotides in length.

In still other embodiments, siNA molecules of the invention comprisesingle-stranded non-circular siNA molecules, wherein the siNA moleculesare independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length.

In various symmetric embodiments, the siNA duplexes of the inventionindependently comprise about 15 to about 30 (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs.Generally, the duplex structure of siNAs of the invention is between 15and 30, more generally between 18 and 25, yet more generally between 19and 24, and most generally between 19 and 21 base pairs in length.

In yet other embodiments, where the duplex siNA molecules of theinvention are asymmetric, the siNA molecules comprise about 3 to 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs. Generally, the duplexstructure of siNAs of the invention is between 15 and 25, more generallybetween 18 and 25, yet more generally between 19 and 24, and mostgenerally between 19 and 21 base pairs in length.

In still other embodiments, where the siNA molecules of the inventionare hairpin or circular structures, the siNA molecules comprise about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs.

The sense strand and antisense strand, or the sense region and antisenseregion, of the siNA molecules of the invention can be complementary.Also, the antisense strand or antisense region can be complementary to anucleotide sequence or a portion thereof of the HBV target RNA. Thesense strand or sense region of the siNA can comprise a nucleotidesequence of a HBV gene or a portion thereof. In certain embodiments, thesense region or sense strand of an siNA molecule of the invention iscomplementary to that portion of the antisense region or antisensestrand of the siNA molecule that is complementary to a HBV targetpolynucleotide sequence, such as for example, but not limited to, thosesequences represented by GENBANK Accession Nos. shown in Table 7.

In some embodiments, siNA molecules of the invention have perfectcomplementarity between the sense strand or sense region and theantisense strand or antisense region of the siNA molecule. In other orthe same embodiments, the antisense strand of the siNA molecules of theinvention are perfectly complementary to a corresponding target nucleicacid molecule.

In yet other embodiments, siNA molecules of the invention have partialcomplementarity (i.e., less than 100% complementarity) between the sensestrand or sense region and the antisense strand or antisense region ofthe siNA molecule or between the antisense strand or antisense region ofthe siNA molecule and a corresponding target nucleic acid molecule.Thus, in some embodiments, the double-stranded nucleic acid molecules ofthe invention, have between about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesin one strand that are complementary to the nucleotides of the otherstrand. In other embodiments, the molecules have between about 15 toabout 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) nucleotides in the sense region that arecomplementary to the nucleotides of the antisense region. of thedouble-stranded nucleic acid molecule. In certain embodiments, thedouble-stranded nucleic acid molecules of the invention have betweenabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) nucleotides in the antisense strand thatare complementary to a nucleotide sequence of its corresponding targetnucleic acid molecule.

In other embodiments, the siNA molecule can contain one or morenucleotide deletions, substitutions, mismatches and/or additions;provided, however, that the siNA molecule maintains its activity, forexample, to mediate RNAi. In a non-limiting example, the deletion,substitution, mismatch and/or addition can result in a loop or bulge, oralternately a wobble or other alternative (non Watson-Crick) base pair.Thus, in some embodiments, for example, the double-stranded nucleic acidmolecules of the invention, have 1 or more (e.g., 1, 2, 3, 4, 5, or 6)nucleotides, in one strand or region that are mismatches ornon-base-paired with the other strand or region. In other embodiments,the double-stranded nucleic acid molecules of the invention, have 1 ormore (e.g., 1, 2, 3, 4, 5, or 6) nucleotides in each strand or regionthat are mismatches or non-base-paired with the other strand or region.In a preferred embodiment, the siNA of the invention contains no morethan 3 mismatches. If the antisense strand of the siNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.

In other embodiments, the siNA molecule can contain one or morenucleotide deletions, substitutions, mismatches and/or additions to asequence in Table 1b provided, however, that the siNA molecule maintainsits activity, for example, to mediate RNAi. In a non-limiting example,the deletion, substitution, mismatch and/or addition can result in aloop or bulge, or alternately a wobble or other alternative (nonWatson-Crick) base pair.

The invention also comprises double-stranded nucleic acid (siNA)molecules as otherwise described hereinabove in which the first strandand second strand are complementary to each other and wherein at leastone strand is hybridisable to the polynucleotide sequence of a sequencein Table 1b under conditions of high stringency, and wherein any of thenucleotides is unmodified or chemically modified.

Hybridization techniques are well known to the skilled artisan (see forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). Preferred stringent hybridization conditions include overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA; followed by washing the filters in0.1×SSC at about 65° C.

In one specific embodiment, the first strand has about 15, 16, 17, 18,19, 20 or 21 nucleotides that are complementary to the nucleotides ofthe other strand and at least one strand is hybridisable to apolynucleotide sequence in Table 1b. In a more preferred embodiment, thefirst strand has about 15, 16, 17, 18, 19, 20 or 21 nucleotides that arecomplementary to the nucleotides of the other strand and at least onestrand is hybridisable to SEQ ID NO: 1, SEQ ID NO: 1210, SEQ ID NO: 2,SEQ ID NO: 1211, SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ IDNO:1213; under conditions of high stringency, and wherein any of thenucleotides is unmodified or chemically modified.

In certain embodiments, the siNA molecules of the invention compriseoverhangs of about 1 to about 4 (e.g., about 1, 2, 3 or 4) nucleotides.The nucleotides in the overhangs can be the same or differentnucleotides. In some embodiments, the overhangs occur at the 3′-end atone or both strands of the double-stranded nucleic acid molecule. Forexample, a double-stranded nucleic acid molecule of the invention cancomprise a nucleotide or non-nucleotide overhang at the 3′-end of theantisense strand/region, the 3′-end of the sense strand/region, or boththe antisense strand/region and the sense strand/region of thedouble-stranded nucleic acid molecule.

In some embodiments, the nucleotides comprising the overhang portion ofan siNA molecule of the invention comprise sequences based on the HBVtarget polynucleotide sequence in which nucleotides comprising theoverhang portion of the antisense strand/region of an siNA molecule ofthe invention can be complementary to nucleotides in the HBV targetpolynucleotide sequence and/or nucleotides comprising the overhangportion of the sense strand/region of an siNA molecule of the inventioncan comprise the nucleotides in the HBV target polynucleotide sequence.Thus, in some embodiments, the overhang comprises a two nucleotideoverhang that is complementary to a portion of the HBV targetpolynucleotide sequence. In other embodiments, however, the overhangcomprises a two nucleotide overhang that is not complementary to aportion of the HBV target polynucleotide sequence. In certainembodiments, the overhang comprises a 3′-UU overhang that is notcomplementary to a portion of the HBV target polynucleotide sequence. Inother embodiments, the overhang comprises a UU overhang at the 3′ end ofthe antisense strand and a TT overhang at the 3′ end of the sensestrand. In other embodiments, the overhang comprises nucleotides asdescribed in the examples, Tables, and Figures herein.

In any of the embodiments of the siNA molecules described herein having3′-terminal nucleotide overhangs, the overhangs are optionallychemically modified at one or more nucleic acid sugar, base, or backbonepositions. Representative, but not limiting examples of modifiednucleotides in the overhang portion of a double-stranded nucleic acid(siNA) molecule of the invention include: 2′-O-alkyl (e.g.,2′-O-methyl), 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino(FANA), 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methylnucleotides. In more preferred embodiments, the overhang nucleotides areeach independently, a 2′-O-alkyl nucleotide, a 2′-O-methyl nucleotide, a2′-dexoy-2-fluoro nucleotide, or a 2′-deoxy ribonucleotide. In someinstances the overhang nucleotides are linked by a one or morephosphorothioate linkages.

In yet other embodiments, siNA molecules of the invention compriseduplex nucleic acid molecules with blunt ends (i.e., without nucleotideoverhangs), where both ends are blunt, or alternatively, where one ofthe ends is blunt. In some embodiments, the siNA molecules of theinvention can comprises one blunt end, for example wherein the 5′-end ofthe antisense strand and the 3′-end of the sense strand do not have anyoverhanging nucleotides. In another example, the siNA molecule comprisesone blunt end, for example wherein the 3′-end of the antisense strandand the 5′-end of the sense strand do not have any overhangingnucleotides. In other embodiments, siNA molecules of the inventioncomprise two blunt ends, for example wherein the 3′-end of the antisensestrand and the 5′-end of the sense strand as well as the 5′-end of theantisense strand and 3′-end of the sense strand do not have anyoverhanging nucleotides.

In any of the embodiments or aspects of the siNA molecules of theinvention, the sense strand and/or the antisense strand can further havea cap, such as described herein or as known in the art, at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of the sense strand and/orantisense strand. Or as in the case of a hairpin siNA molecule, the capcan be at either one or both of the terminal nucleotides of thepolynucleotide. In some embodiments, the cap is at one of both of theends of the sense strand of a double-stranded siNA molecule. In otherembodiments, the cap is at the 3′-end of antisense (guide) strand. Inpreferred embodiments, the caps are at the 3′-end of the sense strandand the 5′-end of the sense strand.

Representative, but non-limiting examples of such terminal caps includean inverted abasic nucleotide, an inverted deoxy abasic nucleotide, aninverted nucleotide moiety, a group shown in FIG. 5, a glycerylmodification, an alkyl or cycloalkyl group, a heterocycle, or any othercap as is generally known in the art.

Any of the embodiments of the siNA molecules of the invention can have a5′ phosphate termini. In some embodiments, the siNA molecules lackterminal phosphates.

Any siNA molecule or construct of the invention can comprise one or morechemical modifications. Modifications can be used to improve in vitro orin vivo characteristics such as stability, activity, toxicity, immuneresponse (e.g., prevent stimulation of an interferon response, aninflammatory or pro-inflammatory cytokine response, or a Toll-likeReceptor (TlF) response), and/or bioavailability.

Applicants describe herein chemically modified siNA molecules withimproved RNAi activity and/or stability compared to correspondingunmodified siNA molecules. Various chemically modified siNA motifsdisclosed herein provide the capacity to maintain RNAi activity that issubstantially similar to unmodified or minimally modified active siRNA(see for example Elbashir et al., 2001, EMBO J., 20:6877-6888) while atthe same time providing nuclease resistance and pharmacokineticproperties suitable for use in therapeutic applications.

In various embodiments, the siNA molecules of the invention comprisemodifications wherein any (e.g., one or more or all) nucleotides presentin the sense and/or antisense strand are modified nucleotides (e.g.,wherein one nucleotide is modified, some nucleotides (i.e., plurality ormore than one) are modified, or all nucleotides are modifiednucleotides. In some embodiments, the siNA molecules of the inventionare partially modified (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, or 59 nucleotides are modified) with chemicalmodifications. In some embodiments, an siNA molecule of the inventioncomprises at least about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60nucleotides that are modified nucleotides. In other embodiments, thesiNA molecules of the invention are completely modified (e.g., 100%modified) with chemical modifications, i.e., the siNA molecule does notcontain any ribonucleotides. In some of embodiments, one or more of thenucleotides in the sense strand of the siNA molecules of the inventionare modified. In the same or other embodiments, one or more of thenucleotides in the antisense strand of the siNA molecules of theinvention are modified.

The chemical modification within a single siNA molecule can be the sameor different. In some embodiments, at least one strand has at least onechemical modification. In other embodiments, each strand has at leastone chemical modifications, which can be the same or different, such as,sugar, base, or backbone (i.e., internucleotide linkage) modifications.In other embodiments, siNA molecules of the invention contain at least2, 3, 4, 5, or more different chemical modifications.

Non-limiting examples of chemical modifications that are suitable foruse in the present invention, are disclosed in U.S. patent applicationSer. Nos. 10/444,853; 10/981,966; 12/064,014 and in references citedtherein and include sugar, base, and phosphate, non-nucleotidemodifications, and/or any combination thereof.

In certain specific embodiments of the invention, at least one modifiednucleotide is a 2′-deoxy-2-fluoro nucleotide, a 2′-deoxy nucleotide, a2′-O-alkyl (e.g., 2′-O-methyl) nucleotide, or a locked nucleic acid(LNA) nucleotide as is generally recognized in the art.

In yet other embodiment of the invention, at least one nucleotide has aribo-like, Northern or A form helix configuration (see e.g., Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).Non-limiting examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoronucleotides; 2′-deoxy-2′-chloro nucleotides; 2′-azido nucleotides;2′-O-trifluoromethyl nucleotides; 2′-O-ethyl-trifluoromethoxynucleotides; 2′-O-difluoromethoxy-ethoxy nucleotides; 4′-thionucleotides and 2′-O-methyl nucleotides.

In various embodiments, a majority (e.g., greater than 50%) of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In some of the same and/or otherembodiments, a majority (e.g., greater than 50%) of the purinenucleotides present in the double-stranded siNA molecule comprises asugar modification.

In some embodiments, the pyrimidine nucleotides in the antisense strandare 2′-O-methyl or 2′-deoxy-2′-fluoro pyrimidine nucleotides and thepurine nucleotides present in the antisense strand are 2′-O-methylnucleotides or 2′-deoxy nucleotides. In other embodiments, thepyrimidine nucleotides in the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the sensestrand are 2′-O-methyl or 2′-deoxy purine nucleotides.

In certain embodiments of the invention, all the pyrimidine nucleotidesin the complementary region on the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides. In certain embodiments, all of the pyrimidinenucleotides in the complementary region of the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides. In certain embodiments, allthe purine nucleotides in the complementary region on the sense strandare 2′-deoxy purine nucleotides. In certain embodiments, all of thepurines in the complementary region on the antisense strand are2′-O-methyl purine nucleotides. In certain embodiments, all of thepyrimidine nucleotides in the complementary regions on the sense strandare 2′-deoxy-2′-fluoro pyrimidine nucleotides; all of the pyrimidinenucleotides in the complementary region of the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides; all the purine nucleotides inthe complementary region on the sense strand are 2′-deoxy purinenucleotides and all of the purines in the complementary region on theantisense strand are 2′-O-methyl purine nucleotides.

In some embodiments, at least 5 or more of the pyrimidine nucleotides inone or both stands are 2′-deoxy-2′-fluoro pyrimidine nucleotides. Insome embodiments, at least 5 or more of the pyrimidine nucleotides inone or both stands are 2′-O-methyl pyrimidine nucleotides. In someembodiments, at least 5 or more of the purine nucleotides in one or bothstands are 2′-deoxy-2′-fluoro purine nucleotides In some embodiments, atleast 5 or more of the purine nucleotides in one or both stands are2′-O-methyl purine nucleotides.

In certain embodiments, the purines and pyrimidines are differentiallymodified at the 2′-sugar position (i.e., at least one purine has adifferent modification from at least one pyrimidine in the same ordifferent strand at the 2′-sugar position). For example, in someinstances, at least 5 or more of the pyrimidine nucleotides in one orboth stands are 2′-deoxy-2′-fluoro pyrimidine nucleotides and at least 5or more purine nucleotides in one or both strands are 2′-O-methyl purinenucleotides. In other instances at least 5 or more of the pyrimidinenucleotides in one or both stands are 2′-O-methyl pyrimidine nucleotidesand at least 5 or more purine nucleotides in one or both strands are2′-deoxy-2′-fluoro purine nucleotides.

Further non-limiting examples of sense and antisense strands of suchsiNA molecules having various modifications and modifications patternsare shown in FIGS. 2 and 3.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of thesiNA molecules of the invention.

The modified siNA molecules of the invention can comprise modificationsat various locations within the siNA molecule. In some embodiments, thedouble-stranded siNA molecule of the invention comprises modifiednucleotides at internal base paired positions within the siNA duplex. Inother embodiments, a double-stranded siNA molecule of the inventioncomprises modified nucleotides at non-base paired or overhang regions ofthe siNA molecule. In yet other embodiments, a double-stranded siNAmolecule of the invention comprises modified nucleotides at terminalpositions of the siNA molecule. For example, such terminal regionsinclude the 3′-position and/or 5′-position of the sense and/or antisensestrand or region of the siNA molecule. Additionally, any of the modifiedsiNA molecules of the invention can have a modification in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. Moreover, with regard tochemical modifications of the siNA molecules of the invention, eachstrand of the double-stranded siNA molecules of the invention can haveone or more chemical modifications, such that each strand comprises adifferent pattern of chemical modifications.

In certain embodiments each strand of a double-stranded siNA molecule ofthe invention comprises a different pattern of chemical modifications,such as any Stab modification chemistries described herein (see Table 8)or any combination thereof, i.e., different combinations of definedStabilization chemistry (Stab) sense and antisense strands. Further,non-limiting examples of modification schemes that could give rise todifferent patterns of modifications are shown in Table 8. Thestabilization chemistries referred to in Table 8 as Stab, can becombined in any combination of sense/antisense chemistries, such as Stab7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13,Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab18/20, Stab 7/32, Stab 8/32, or Stab 18/32 or any other combination ofStabilization chemistries.

In any of the siNAs of the invention, one or more (for example 1, 2, 3,4 or 5) nucleotides at the 5′-end of the guide strand or guide region(also known as antisense strand or antisense region) of the siNAmolecule are ribonucleotides.

In certain embodiments, the present invention provides a double-strandedshort interfering nucleic acid (siNA) molecule that modulates theexpression of HBV, wherein the siNA comprises a sense strand and anantisense strand; each strand is independently 15 to 30 nucleotides inlength; and the antisense strand comprises at least 15 nucleotideshaving sequence complementary to any of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′; (SEQ ID NO: 2)5′-GUGGUGGACUUCUCUCAAU-3′; (SEQ ID NO: 3) 5′-GCCGAUCCAUACUGCGGAA-3′; or(SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′.

In some embodiments, the antisense strand of a siNA molecule of theinvention comprises at least a 15 nucleotide sequence of:

(SEQ ID NO: 452) 5′-UGAGAGAAGUCCACCACGA-3′; (SEQ ID NO: 453)5′-AUUGAGAGAAGUCCACCAC-3′; (SEQ ID NO: 454) 5′-UUCCGCAGUAUGGAUCGGC-3′;or (SEQ ID NO: 455) 5′-GUUCCGCAGUAUGGAUCGG-3′.

In some embodiments, the sense strand of a siNA molecule of theinvention comprises at least a 15 nucleotide sequence of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′; (SEQ ID NO: 2)5′-GUGGUGGACUUCUCUCAAU-3′; (SEQ ID NO: 3) 5′-GCCGAUCCAUACUGCGGAA-3′; or(SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′.

In some embodiments, a siNA molecule of the invention comprises any of:

(SEQ ID NO: 1) 5′-UCGUGGUGGACUUCUCUCA-3′ and (SEQ ID NO: 452)5′-UGAGAGAAGUCCACCACGA-3′; or (SEQ ID NO: 2) 5′-GUGGUGGACUUCUCUCAAU-3′;and (SEQ ID NO: 453) 5′-AUUGAGAGAAGUCCACCAC-3′; or (SEQ ID NO: 3)5′-GCCGAUCCAUACUGCGGAA-3′ and (SEQ ID NO: 454)5′-UUCCGCAGUAUGGAUCGGC-3′; or (SEQ ID NO: 4) 5′-CCGAUCCAUACUGCGGAAC-3′;and (SEQ ID NO: 455) 5′-GUUCCGCAGUAUGGAUCGG-3′.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of theseembodiments.

In certain embodiments, the nucleotides of the at least a 15 nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ ID NO: 2, SEQ ID NO: 453,SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, or SEQ ID NO:455 form acontiguous stretch of nucleotides.

In some embodiments, the siNA molecule can contain one or morenucleotide deletions, substitutions, mismatches and/or additions to theat least 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ IDNO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, orSEQ ID NO:455; provided, however, that the siNA molecule maintains itsactivity, for example, to mediate RNAi. In a non-limiting example, thedeletion, substitution, mismatch and/or addition can result in a loop orbulge, or alternately a wobble or other alternative (non Watson-Crick)base pair.

In certain embodiments of the invention, double-stranded siNA moleculesare provided, wherein the molecule has a sense strand and an antisensestrand and comprises the following formula (A):B—N_(X3)(N)_(X2)—(N)_(X2)B-3′B(N)_(X1)—N_(X4)—[N]_(X5)-5′  (A)

-   -   wherein, the upper strand is the sense strand and the lower        strand is the antisense strand of the double-stranded nucleic        acid molecule; wherein the antisense strand comprises at least a        15 nucleotide sequence of SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID        NO: 454, or SEQ ID NO: 455, and the sense strand comprises a        sequence having complementarity to the antisense strand;    -   each N is independently a nucleotide which is unmodified or        chemically modified or a non-nucleotide;    -   each B is a terminal cap that is present or absent;    -   (N) represents overhanging nucleotides, each of which is        independently unmodified or chemically modified;    -   [N] represents nucleotides that are ribonucleotides;    -   X1 and X2 are independently integers from 0 to 4;    -   X3 is an integer from 15 to 30;    -   X4 is an integer from 9 to 30; and    -   X5 is an integer from 0 to 6, provided that the sum of X4 and X5        is 15-30.

In certain embodiments, the nucleotides of the at least a 15 nucleotidesequence of SEQ ID NO: 452, SEQ ID NO: 453, SEQ ID NO: 454, or SEQ IDNO: 455 form a contiguous stretch of nucleotides.

In some embodiments, the siNA molecule of formula A can contain one ormore nucleotide deletions, substitutions, mismatches and/or additions tothe at least 15 nucleotide sequence of SEQ ID NO: 452, SEQ ID NO: 453,SEQ ID NO: 454, or SEQ ID NO: 455; provided, however, that the siNAmolecule maintains its activity, for example, to mediate RNAi. In anon-limiting example, the deletion, substitution, mismatch and/oraddition can result in a loop or bulge, or alternately a wobble or otheralternative (non Watson-Crick) base pair.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) of formula (A); wherein

-   -   (a) one or more pyrimidine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   (b) one or more purine nucleotides in N_(X4) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof;    -   (c) one or more pyrimidine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof; and    -   (d) one or more purine nucleotides in N_(X3) positions are        independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl        nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any        combination thereof.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A); wherein

-   -   (a) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X4)        positions are 2′-deoxy-2′-fluoro nucleotides;    -   (b) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X4) positions        are 2′-O-alkyl nucleotides;    -   (c) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X3)        positions are 2′-deoxy-2′-fluoro nucleotides; and    -   (d) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X3) positions        are 2′-deoxy nucleotides.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A); wherein

-   -   (a) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X4)        positions are 2′-O-alkyl nucleotides;    -   (b) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X4) positions        are ribonucleotides;    -   (c) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X3)        positions are 2′-O-alkyl nucleotides; and    -   (d) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X3) positions        are ribonucleotides.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A); wherein

-   -   (a) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X4)        positions are 2′-deoxy-2′-fluoro nucleotides;    -   (b) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X4) positions        are 2′-O-alkyl nucleotides;    -   (c) 1, 2, 3, 4, 5 or more pyrimidine nucleotides in N_(X3)        positions are 2′-O-alkyl nucleotides; and    -   (d) 1, 2, 3, 4, 5 or more purine nucleotides in N_(X3) positions        are 2′-deoxy-2′-fluoro nucleotides.

In certain embodiments, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule of formula (A) furthercomprising one or more phosphorothioate internucleotide linkages.

In some embodiments, siNA molecules having formula A comprise a terminalphosphate group at the 5′-end of the antisense strand or antisenseregion of the nucleic acid molecule.

In various embodiments, siNA molecules having formula A comprise X5=0,1, 2, or 3; each X1 and X2=1 or 2; X3=18, 19, 20, 21, 22, or 23, andX4=17, 18, 19, 20, 21, 22, or 23.

In certain embodiments, siNA molecules having formula A comprise X5=3.In other embodiments siNA molecules having formula A comprise X5=0.

In certain embodiments, siNA molecules having formula A comprise X1=2and X2=2.

In various embodiments, siNA molecules having formula A comprise X5=0,X1=2, and X2=2. In other embodiments, siNA molecules having formula Acomprise X5=3, X1=2, and X2=2.

In one specific embodiment, an siNA molecule having formula A comprisesX5=3; each X1 and X2=2; X3=19, and X4=16.

In another specific embodiment, an siNA molecule having formula Acomprises X5=0; each X1 and X2=2; X3=19, and X4=19.

In certain embodiments, siNA molecules having formula A comprise caps(B) at the 3′ and 5′ ends of the sense strand or sense region.

In certain embodiments, siNA molecules having formula A comprise caps(B) at the 3′-end of the antisense strand or antisense region.

In various embodiments, siNA molecules having formula A comprise caps(B) at the 3′ and 5′ ends of the sense strand or sense region and caps(B) at the 3′-end of the antisense strand or antisense region.

In yet other embodiments, siNA molecules having formula A comprise caps(B) only at the 5′-end of the sense (upper) strand of thedouble-stranded nucleic acid molecule.

In some embodiments, siNA molecules having formula A further compriseone or more phosphorothioate internucleotide linkages between thenucleotides. In certain embodiments, siNA molecules having formula Acomprise one or more phosphorothioate internucleotide linkages betweenthe first terminal (N) and the adjacent nucleotide on the 3′ end of thesense strand, antisense strand, or both sense strand and antisensestrands of the nucleic acid molecule. For example, a double-strandednucleic acid molecule can comprise X1 and/or X2=2 having overhangingnucleotide positions with a phosphorothioate internucleotide linkage,e.g., (NsN) where “s” indicates phosphorothioate.

In some embodiments, one or more of the nucleotides of siNA moleculeshaving formula A have a universal base.

In certain embodiments, siNA molecules having formula A have at position14 from the 5′-end of the antisense strand a ribonucleotide when thenucleotide at that position 14 is a purine. In other embodiments, siNAmolecules having formula A have at position 14 from the 5′-end of theantisense strand a ribonucleotide, a 2′-deoxy-2′-fluoro nucleotide or a2′-O-methyl nucleotide when the nucleotide at that position 14 is apyrimidine nucleotide.

In some embodiments, siNA molecules having formula A comprises (N)nucleotides in the antisense strand (lower strand) that arecomplementary to nucleotides in a HBV target polynucleotide sequence,which also has complementarity to the N and [N] nucleotides of theantisense (lower) strand.

Any of the above described modifications, or combinations thereof,discussed above as applicable to siNAs of the invention, including thosein the references cited, can be applied to any of the embodiments tosiNA molecules having formula A.

C. Generation/Synthesis of siNA Molecules

The siNAs of the invention can be obtained using a number of techniquesknown to those of skill in the art. For example the siNA can bechemically synthesized or may be encoded by plasmid (e.g., transcribedas sequences that automatically fold into duplexes with hairpin loops).siNA can also be generated by cleavage of longer dsRNA (e.g., dsRNAgreater than about 25 nucleotides in length) by the E coli RNase II orDicer. These enzymes process the dsRNA into biologically active siNA(see, e.g., Yang et al., PNAS USA 99:9942-9947 (2002); Calegari et al.PNAS USA 99:14236 (2002) Byron et al. Ambion Tech Notes; 10 (1):4-6(2009); Kawaski et al., Nucleic Acids Res., 31:981-987 (2003), Knightand Bass, Science, 293:2269-2271 (2001) and Roberston et al., J. Biol.Chem 243:82 (1969).

1. Chemical Synthesis

Preferably, siNA of the invention are chemically synthesized.Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.

siNA molecules without modifications are synthesized using procedures asdescribed in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, Nucleic Acids Res., 18, 5433. These syntheses makes use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end thatcan be used for certain siNA molecules of the invention.

In certain embodiments, the siNA molecules of the invention aresynthesized, deprotected, and analyzed according to methods described inU.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442,and U.S. patent application Ser. No. 10/190,359.

In a non-limiting synthesis example, small scale syntheses are conductedon a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scaleprotocol with a 2.5 min coupling step for 2′-O-methylated nucleotidesand a 45 second coupling step for 2′-deoxy nucleotides or2′-deoxy-2′-fluoro nucleotides. Table 9 outlines the amounts and thecontact times of the reagents used in the synthesis cycle.

Alternatively, the siNA molecules of the present invention can besynthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT Publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

Various siNA molecules of the invention can also be synthesized usingthe teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400;and 6,111,086.

2. Vector Expression

Alternatively, siNA molecules of the invention that interact with anddown-regulate gene encoding target HBV molecules can be expressed anddelivered from transcription units (see for example Couture et al.,1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinantvectors can be DNA plasmids or viral vectors. siNA expressing viralvectors can be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, or alphavirus.

In some embodiments, pol III based constructs are used to expressnucleic acid molecules of the invention. Transcription of the siNAmolecule sequences can be driven from a promoter for eukaryotic RNApolymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III(pol III). (see for example, Thompson, U.S. Pat. Nos. 5,902,880 and6,146,886). (See also, Izant and Weintraub, 1985, Science, 229, 345;McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399;Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5;Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic etal., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol.,65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver etal., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic AcidsRes., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Transcripts frompol II or pol III promoters are expressed at high levels in all cells;the levels of a given pol II promoter in a given cell type depends onthe nature of the gene regulatory sequences (enhancers, silencers, etc.)present nearby. Prokaryotic RNA polymerase promoters are also used,providing that the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72;Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990,Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstratedthat nucleic acid molecules expressed from such promoters can functionin mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res.Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al.,1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992,EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci.U.S.A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259;Sullenger & Cech, 1993, Science, 262, 1566). More specifically,transcription units such as the ones derived from genes encoding U6small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA areuseful in generating high concentrations of desired RNA molecules suchas siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996,supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg etal., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45;Beigelman et al., International PCT Publication No. WO 96/18736. Theabove siNA transcription units can be incorporated into a variety ofvectors for introduction into mammalian cells, including but notrestricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated virus vectors), or viral RNA vectors(such as retroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

Vectors used to express the siNA molecules of the invention can encodeone or both strands of an siNA duplex, or a single self-complementarystrand that self hybridizes into an siNA duplex. The nucleic acidsequences encoding the siNA molecules of the instant invention can beoperably linked in a manner that allows expression of the siNA molecule(see for example Paul et al., 2002, Nature Biotechnology, 19, 505;Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al.,2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, NatureMedicine, advance online publication doi:10.1038/nm725).

D. Carrier/Delivery Systems

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or as arecombinant plasmid or viral vectors which express the siNA molecules,or otherwise delivered to target cells or tissues. Methods for thedelivery of nucleic acid molecules are described in Akhtar et al., 1992,Trends Cell Bio., 2, 139; Delivery Strategies for AntisenseOligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999,Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752,184-192. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al.,PCT WO 94/02595 further describe the general methods for delivery ofnucleic acid molecules. These protocols can be utilized for the deliveryof virtually any nucleic acid molecule. Nucleic acid molecules can beadministered to cells by a variety of methods known to those of skill inthe art, including, but not restricted to, encapsulation in liposomes,by iontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for example,Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT Publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722).

In one aspect, the present invention provides carrier systems containingthe siNA molecules described herein. In some embodiments, the carriersystem is a lipid-based carrier system, cationic lipid, or liposomenucleic acid complexes, a liposome, a micelle, a virosome, a lipidnanoparticle or a mixture thereof. In other embodiments, the carriersystem is a polymer-based carrier system such as a cationicpolymer-nucleic acid complex. In additional embodiments, the carriersystem is a cyclodextrin-based carrier system such as a cyclodextrinpolymer-nucleic acid complex. In further embodiments, the carrier systemis a protein-based carrier system such as a cationic peptide-nucleicacid complex. Preferably, the carrier system is a lipid nanoparticle(“LNP”) formulation.

In certain embodiments, the siNA molecules of the invention areformulated with a lipid nanoparticle composition such as is described inU.S. patent application Ser. Nos. 11/353,630, 11/586,102, 61/189,295,61/204,878, 61/235,476, 61/249,807, 61/298,022, 61/351,373, 61/347,640,61/345,754, 61/322,054, 12/640,342, and 12/617,079, and PCT ApplicationsNos. PCT/US10/020013 and PCT/US09/053336. In certain embodiments, thesiNA is formulated with components and ratios described and exemplifiedin Example 6 herein (see also Tables 10 & 11).

In various embodiments, lipid nanoparticle formulations described inTable 10 are applied to any siNA molecule or combination of siNAmolecules herein. In some embodiments, the invention features acomposition comprising an siNA molecule of the invention formulated as acomposition including any of cationic lipid compound numbers 1-46 or acombination thereof.

In certain other embodiments, the invention features a compositioncomprising an siNA molecule of the invention formulated with any of thecationic lipid formulations described in U.S. Patent Application Nos.61/189,295, 61/204,878, 61/235,476, 61/249,807, and 61/298,022.

In other embodiments, the invention features conjugates and/or complexesof siNA molecules of the invention. Such conjugates and/or complexes canbe used to facilitate delivery of siNA molecules into a biologicalsystem, such as a cell. The conjugates and complexes provided by theinstant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. Non-limiting, examples of such conjugatesare described in U.S. Publication Nos. US2008/0152661 A1 and US2004/0162260 A1 (e.g., CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.) andU.S. patent application Ser. Nos. 10/427,160 10/201,394, 61/322422, and61/315,223; and U.S. Pat. Nos. 6,528,631; 6,335,434; 6,235,886;6,153,737; 5,214,136; and 5,138,045.

In various embodiments, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 100 to about 50,000daltons (Da).

In yet other embodiments, the invention features compositions orformulations comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes orstealth liposomes) and siNA molecules of the invention, such as isdisclosed in for example, International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392.

In some embodiments, the siNA molecules of the invention can also beformulated or complexed with polyethyleneimine and derivatives thereof,such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine(PEI-PEG-GAL) orpolyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in U.S. PatentApplication Publication No. 20030077829.

In other embodiments, siNA molecules of the invention are complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666. In still other embodiments, themembrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310.

In certain embodiments, siNA molecules of the invention are complexedwith delivery systems as described in U.S. Patent ApplicationPublication Nos. 2003077829; 20050287551; 20050164220; 20050191627;20050118594; 20050153919; 20050085486; and 20030158133; andInternational PCT Publication Nos. WO 00/03683 and WO 02/087541.

In some embodiments, a liposomal formulation of the invention comprisesan siNA molecule of the invention (e.g., siNA) formulated or complexedwith compounds and compositions described in U.S. Pat. Nos. 6,858,224;6,534,484; 6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432;6,586,001; 6,120,798; 6,977,223; 6,998,115; 5,981,501; 5,976,567;5,705,385; and U.S. Patent Application Publication Nos. 2006/0019912;2006/0019258; 2006/0008909; 2005/0255153; 2005/0079212; 2005/0008689;2003/0077829, 2005/0064595, 2005/0175682, 2005/0118253; 2004/0071654;2005/0244504; 2005/0265961 and 2003/0077829.

Alternatively, recombinant plasmids and viral vectors, as discussedabove, which express siNAs of the invention can be used to deliver themolecules of the invention. Delivery of siNA molecule expressing vectorscan be systemic, such as by intravenous or intra-muscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell(for a review see Couture et al., 1996, TIG., 12, 510). Such recombinantplasmids can also be administered directly or in conjunction with asuitable delivery reagents, including, for example, the Mirus TransitLT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin;polycations (e.g., polylysine) or liposomes lipid-based carrier system,cationic lipid, or liposome nucleic acid complexes, a micelle, avirosome, a lipid nanoparticle.

E. Kits

The present invention also provides nucleic acids in kit form. The kitmay comprise a container. The kit typically contains a nucleic acid ofthe invention with instructions for its administration. In certaininstances, the nucleic acids may have a targeting moiety attached.Methods of attaching targeting moieties (e.g. antibodies, proteins) areknown to those of skill in the art. In certain instances, the nucleicacids are chemically modified. In other embodiments, the kit containsmore than one siNA molecule of the invention. The kits may comprise ansiNA molecule of the invention with a pharmaceutically acceptablecarrier or diluent. The kits may further comprise excipients.

F. Therapeutic Uses/Pharmaceutical Compositions

The present body of knowledge in HBV research indicates the need formethods to assay HBV activity and for compounds that can regulate HBVexpression for research, diagnostic, and therapeutic use. As describedinfra, the nucleic acid molecules of the present invention can be usedin assays to diagnose disease state related of HBV levels. In addition,the nucleic acid molecules and pharmaceutical compositions can be usedto treat disease states related to HBV RNA levels.

1. Disease States Associated with HBV

Particular disease states that can be associated with HBV expressionmodulation include liver disease and cancer. Non-limiting examples ofsuch diseases include cirrhosis of the liver and heptocellularcarcinoma.

It is understood that the siNA molecules of the invention can degradethe target HBV mRNA (and thus inhibit the diseases stated above).Inhibition of a disease can be evaluated by directly measuring theprogress of the disease in a subject. It can also be inferred throughobserving a change or reversal in a condition associated with thedisease. Additionally, the siNA molecules of the invention can be usedas a prophylaxis. Thus, the use of the nucleic acid molecules andpharmaceutical compositions of the invention can be used to ameliorate,treat, prevent, and/or cure these diseases and others associated withregulation of HBV gene expression.

2. Pharmaceutical Compositions

The siNA molecules of the instant invention provide useful reagents andmethods for a variety of therapeutic, prophylactic, cosmetic,veterinary, diagnostic, target validation, genomic discovery, geneticengineering, and pharmacogenomic applications.

a. Formulations

Thus, the present invention, in one aspect, also provides forpharmaceutical compositions of the siNA molecules described, i.e.,compositions in a pharmaceutically acceptable carrier or diluent. Thesepharmaceutical compositions include salts, esters, or salts of suchesters, of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, hydroiodic, acetic acid, and benzenesulfonic acid. Other salts include for example, sodium, potassium,manganese, ammonium, and calcium salts. These formulations orcompositions can comprise a pharmaceutically acceptable carrier ordiluent as is generally known in the art.

In one embodiment, the invention features a pharmaceutical compositioncomprising an siNA molecule comprising at least a 15 nucleotide sequenceof SEQ ID NO: 1. In another embodiment, the invention features apharmaceutical composition comprising an siNA molecule comprising atleast a 15 nucleotide sequence of SEQ ID NO: 452. In yet anotherembodiment, the invention features a pharmaceutical compositioncomprising an siNA molecule comprising at least a 15 nucleotide sequenceof SEQ ID NO: 2. In still another embodiment, the invention features apharmaceutical composition comprising an siNA molecule comprising atleast a 15 nucleotide sequence of SEQ ID NO: 453. In another embodiment,the invention features a pharmaceutical composition comprising an siNAmolecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 3.In another embodiment, the invention features a pharmaceuticalcomposition comprising an siNA molecule comprising at least a 15nucleotide sequence of SEQ ID NO: 454. In another embodiment, theinvention features a pharmaceutical composition comprising an siNAmolecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 4.In yet another embodiment, the invention features a pharmaceuticalcomposition comprising an siNA molecule comprising at least a 15nucleotide sequence of SEQ ID NO: 455. In still another embodiment, theinvention features a pharmaceutical composition comprising an siNAmolecule comprising formula (A).

The siNA molecules of the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art for example as described inRemington's Pharmaceutical Science, 17^(th) ed., Mack PublishingCompany, Easton, Pa. (1985).

In some embodiments, pharmaceutical compositions of the invention (e.g.siNA and/or LNP formulations thereof) further comprise conventionalpharmaceutical excipients and/or additives. Suitable pharmaceuticalexcipients include preservatives, flavoring agents, stabilizers,antioxidants, osmolality adjusting agents, buffers, and pH adjustingagents. Suitable additives include physiologically biocompatible buffers(e.g., trimethylamine hydrochloride), addition of chelants (such as, forexample, DTPA or DTPA-bisamide) or calcium chelate complexes (as forexample calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions ofcalcium or sodium salts (for example, calcium chloride, calciumascorbate, calcium gluconate or calcium lactate). In addition,antioxidants and suspending agents can be used.

Non-limiting examples of various types of formulations for localadministration include ointments, lotions, creams, gels, foams,preparations for delivery by transdermal patches, powders, sprays,aerosols, capsules or cartridges for use in an inhaler or insufflator ordrops (for example eye or nose drops), solutions/suspensions fornebulization, suppositories, pessaries, retention enemas and chewable orsuckable tablets or pellets (for example for the treatment of aphthousulcers) or liposome or microencapsulation preparations.

Ointments, creams and gels, can, for example, can be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Non limiting examples of such bases canthus, for example, include water and/or an oil such as liquid paraffinor a vegetable oil such as arachis oil or castor oil, or a solvent suchas polyethylene glycol. Various thickening agents and gelling agents canbe used depending on the nature of the base. Non-limiting examples ofsuch agents include soft paraffin, aluminum stearate, cetostearylalcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethyleneand cellulose derivatives, and/or glyceryl monostearate and/or non-ionicemulsifying agents.

In one embodiment lotions can be formulated with an aqueous or oily baseand will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents or thickeningagents.

In one embodiment powders for external application can be formed withthe aid of any suitable powder base, for example, talc, lactose orstarch. Drops can be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents,suspending agents or preservatives.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate; or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

In other embodiments, the siNA and LNP compositions and formulationsprovided herein for use in pulmonary delivery further comprise one ormore surfactants. Suitable surfactants or surfactant components forenhancing the uptake of the compositions of the invention includesynthetic and natural as well as full and truncated forms of surfactantprotein A, surfactant protein B, surfactant protein C, surfactantprotein D and surfactant Protein E, di-saturated phosphatidylcholine(other than dipalmitoyl), dipalmitoylphosphatidylcholine,phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol,phosphatidylethanolamine, phosphatidylserine; phosphatidic acid,ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, cholinephosphate; as well as natural and artificial lamellar bodies which arethe natural carrier vehicles for the components of surfactant, omega-3fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid,non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants can be usedeither as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the nucleic acid component of a pharmaceutical composition herein.

b. Combinations

The siNAs and pharmaceutical formulations according to the invention canbe administered to a subject alone or used in combination with orinclude one or more other therapeutic agents, for example, antiviral oranticancer agents. Thus, combinations of the presently disclosedcompounds with other antiviral or anti-cancer or chemotherapeutic agentsare within the scope of the invention

Examples of anti-cancer or chemotherapeutic agents can be found inCancer Principles and Practice of Oncology by V. T. Devita and S.Hellman (editors), 6^(th) edition (Feb. 15, 2001), Lippincott Williams &Wilkins Publishers. A person of ordinary skill in the art would be ableto discern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Suchanti-cancer agents include, but are not limited to, the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic/cytostatic agents, antiproliferativeagents, prenyl-protein transferase inhibitors, HMG-CoA reductaseinhibitors and other angiogenesis inhibitors, inhibitors of cellproliferation and survival signaling, apoptosis inducing agents andagents that interfere with cell cycle checkpoints. The siNAs of theinvention are also useful in combination with any therapeutic agent usedin the treatment of HCC, for example, but not limitation sorafenib.

In a further embodiment, therefore, the invention provides a combinationcomprising an siNA molecule of the invention, such as for example, butnot limitation, an siNA molecule comprising at least a 15 nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 1210, SEQ ID NO: 2, SEQ ID NO:1211, SEQ ID NO: 3, SEQ ID NO: 1212, SEQ ID NO: 4, or SEQ ID NO:1213; orformula (A) or a pharmaceutically acceptable salt, solvate orphysiologically functional derivative thereof together with one or moreantiviral agents. In some embodiments, the antiviral is another HBVinhibitor.

In certain embodiments, the instant siNA molecules of the invention areuseful in combination with known HBV antiviral agents including thefollowing: Lamivudine (2′,3′-dideoxy-3′-thiacytidine, commonly called3TC), PegInterferon-alpha2a, tenofovir, entecavir, telbivudine,adefovir.

Also, the invention provides a combination comprising an siNA moleculeof the invention, such as for example, but not limitation, an siNAmolecule comprising at least a 15 nucleotide sequence of SEQ ID NO: 1,SEQ ID NO: 452, SEQ ID NO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO:454, SEQ ID NO: 4, or SEQ ID NO:455; or formula (A) or apharmaceutically acceptable salt, solvate or physiologically functionalderivative thereof together with one or more anti-cancer orchemotherapeutic agents.

In certain embodiments, the instant siNA molecules of the invention areuseful in combination with known anti-cancer agents including thefollowing: estrogen receptor modulators, androgen receptor modulators,retinoid receptor modulators, cytotoxic agents, antiproliferativeagents, prenyl-protein transferase inhibitors, HMG-CoA reductaseinhibitors, HIV protease inhibitors, reverse transcriptase inhibitors,and other angiogenesis inhibitors.

Examples of estrogen receptor modulators that can be used in combinationwith the compounds of the invention include, but are not limited to,tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene,fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

Examples of androgen receptor modulators that can be used in combinationwith the compounds of the invention include, but are not limited to,finasteride and other 5α-reductase inhibitors, nilutamide, flutamide,bicalutamide, liarozole, and abiraterone acetate.

Examples of such retinoid receptor modulators that can be used incombination with the compounds of the invention include, but are notlimited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, α-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

Examples of cytotoxic agents that can be used in combination with thecompounds of the invention include, but are not limited to, sertenef,cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound that can be used incombination with the compounds of the invention is tirapazamine.

Examples of proteasome inhibitors that can be used in combination withthe compounds of the invention include, but are not limited to,lactacystin and bortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agents thatcan be used in combination with the compounds of the invention include,but are not limited to, paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, are topotecan, hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroOxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis [(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, that can be used in combination with the compoundsof the invention include, but are not limited to, inhibitors describedin PCT Publications WO 01/30768, WO 01/98278, WO 03/050,064, WO03/050,122, WO 03/049,527, WO 03/049,679, WO 03/049,678, WO04/039774,WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171,WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206,WO05/019205, WO05/018547, WO05/017190, US2005/0176776. In an embodimentinhibitors of mitotic kinesins include, but are not limited toinhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitorsof MCAK, inhibitors of Kif14, inhibitors of Mphosph1 and inhibitors ofRab6-KIFL.

Examples of “histone deacetylase inhibitors” that can be used incombination with the compounds of the invention include, but are notlimited to, TSA, oxamflatin, PXD101, MG98, valproic acid and scriptaid.Further reference to other histone deacetylase inhibitors may be foundin the following manuscript; Miller, T. A. et al. J. Med. Chem.46(24):5097-5116 (2003).

Inhibitors of kinases involved in mitotic progression that can be usedin combination with the compounds of the invention include, but are notlimited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases(PLK) (in particular inhibitors of PLK-1), inhibitors of bub-1 andinhibitors of bub-R1.

Antiproliferative agents that can be used in combination with thecompounds of the invention include, but are not limited to, antisenseRNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231,and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur,pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine,galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate,raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed,pemetrexed, nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents that can beused in combination with the compounds of the invention include thosetherapeutic agents which have cytotoxic agents or radioisotopes attachedto a cancer cell specific or target cell specific monoclonal antibody,such as, for example, Bexxar.

Examples of HMG-CoA reductase inhibitors that may be used that can beused in combination with the compounds of the invention include, but arenot limited to, lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938,4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos.4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S.Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589),fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772, 4,911,165,4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896) andatorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893,5,489,691 and 5,342,952). The structural formulas of these andadditional HMG-CoA reductase inhibitors that may be used in the instantmethods are described at page 87 of M. Yalpani, “Cholesterol LoweringDrugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos.4,782,084 and 4,885,314.

Examples of prenyl-protein transferase inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, can be found in the following publications and patents: WO96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO98/28980, WO 98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430,5,532,359, 5,510,510, 5,589,485, 5,602,098, European Patent Publ. 0 618221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181,European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917,WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example ofthe role of a prenyl-protein transferase inhibitor on angiogenesis seeEuropean J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

Examples of angiogenesis inhibitors that can be used in combination withthe compounds of the invention include, but are not limited to, tyrosinekinase inhibitors, such as inhibitors of the tyrosine kinase receptorsFlt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxy-genase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),steroidal anti-inflammatories (such as corticosteroids,mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred,betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, NatureBiotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis may alsobe used in combination with the compounds of the instant invention andinclude agents that modulate or inhibit the coagulation and fibrinolysissystems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examplesof such agents that modulate or inhibit the coagulation and fibrinolysispathways that can be used in combination with the compounds of theinvention include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparins and carboxypeptidase Uinhibitors (also known as inhibitors of active thrombin activatablefibrinolysis inhibitor[TAFIa]) (see Thrombosis Res. 101:329-354 (2001)).TAFIa inhibitors have been described in PCT Publication WO 03/013,526and U.S. Ser. No. 60/349,925 (filed Jan. 18, 2002).

Agents that interfere with cell cycle checkpoints that can be used incombination with the compounds of the invention include, but are notlimited to, inhibitors of ATR, ATM, the Chk1 and Chk2 kinases and cdkand cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

Agents that interfere with receptor tyrosine kinases (RTKs) that can beused in combination with the compounds of the invention include, but arenot limited to, inhibitors of c-Kit, Eph, PDGF, Flt3 and HBV. Furtheragents include inhibitors of RTKs as described by Bume-Jensen andHunter, Nature, 411:355-365, 2001.

Inhibitors of cell proliferation and survival signaling pathway that canbe used in combination with the compounds of the invention include, butare not limited to, inhibitors of EGFR (for example gefitinib anderlotinib), inhibitors of ERB-2 (for example trastuzumab), inhibitors ofIGFR, inhibitors of cytokine receptors, inhibitors of HBV, inhibitors ofPI3K (for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in WO 02/083064, WO02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360,WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO2004/096130, WO 2005/100356, WO 2005/100344), inhibitors of Raf kinase(for example BAY-43-9006), inhibitors of MEK (for example CI-1040 andPD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

Apoptosis inducing agents that can be used in combination with thecompounds of the invention include, but are not limited to, activatorsof TNF receptor family members (including the TRAIL receptors).

NSAIDs that are selective COX-2 inhibitors that can be used incombination with the compounds of the invention include, but are notlimited to, those NSAIDs disclosed in U.S. Pat. Nos. 5,474,995,5,861,419, 6,001,843, 6,020,343, 5,409,944, 5,436,265, 5,536,752,5,550,142, 5,604,260, 5,698,584, 5,710,140, WO 94/15932, U.S. Pat. Nos.5,344,991, 5,134,142, 5,380,738, 5,393,790, 5,466,823, 5,633,272, and5,932,598, all of which are hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in combination with thecompounds of the invention include:3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and5-chloro-3-(4-methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridine;or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Angiogenesis inhibitors that can be used in combination with thecompounds of the invention include, but are not limited to, endostatin,ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]—1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)-phenyl]methyl]—1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

Tyrosine kinase inhibitors that can be used in combination with thecompounds of the invention include, but are not limited to,N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one,SH268, genistein, imatinib (STI571), CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant compositions and methods. For example,combinations of the instantly claimed compounds with PPAR-γ (i.e.,PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are usefulin the treatment of certain malignancies. PPAR-γ and PPAR-δ are thenuclear peroxisome proliferator-activated receptors γ and δ. Theexpression of PPAR-γ on endothelial cells and its involvement inangiogenesis has been reported in the literature (see J. Cardiovasc.Pharmacol. 31:909-913 (1998); J. Biol. Chem. 274:9116-9121 (1999);Invest. Ophthalmol Vis. Sci. 41:2309-2317 (2000)). More recently, PPAR-γagonists have been shown to inhibit the angiogenic response to VEGF invitro; both troglitazone and rosiglitazone maleate inhibit thedevelopment of retinal neovascularization in mice. (Arch. Ophthamol.119:709-717 (2001)). Examples of PPAR-γ agonists and PPAR-γ/α agoniststhat can be used in combination with the compounds of the inventioninclude, but are not limited to, thiazolidinediones (such as DRF2725,CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate,gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555,GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570,PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in U.S.Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of the presentlydisclosed compounds in combination with gene therapy for the treatmentof cancer. For an overview of genetic strategies to treating cancer seeHall et al. (Am J Hum Genet 61:785-789 (1997)) and Kufe et al. (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton, 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 5(8):1105-13 (1998)), andinterferon gamma (J Immunol 164:217-222 (2000)).

The compounds of the instant invention may also be administered incombination with an inhibitor of inherent multidrug resistance (MDR), inparticular MDR associated with high levels of expression of transporterproteins. Such MDR inhibitors include inhibitors of p-glycoprotein(P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833(valspodar).

A compound of the present invention may be employed in conjunction withanti-emetic agents to treat nausea or emesis, including acute, delayed,late-phase, and anticipatory emesis, which may result from the use of acompound of the present invention, alone or with radiation therapy. Forthe prevention or treatment of emesis, a compound of the presentinvention may be used in conjunction with other anti-emetic agents,especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists,such as ondansetron, granisetron, tropisetron, and zatisetron, GABABreceptor agonists, such as baclofen, a corticosteroid such as Decadron(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten orothers such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401,3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, anantidopaminergic, such as the phenothiazines (for exampleprochlorperazine, fluphenazine, thioridazine and mesoridazine),metoclopramide or dronabinol. In an embodiment, an anti-emesis agentselected from a neurokinin-1 receptor antagonist, a 5HT3 receptorantagonist and a corticosteroid is administered as an adjuvant for thetreatment or prevention of emesis that may result upon administration ofthe instant compounds.

Neurokinin-1 receptor antagonists of use in conjunction with thecompounds of the present invention are fully described, for example, inU.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595,5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European PatentPublication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733632 and 0 776 893; PCT International Patent Publication Nos. WO90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151,92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116,93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181,93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429,94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165,94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767,94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309,95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549,95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129,95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094,96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304,96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553,97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084,97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529,2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293169, and 2 302 689. The preparation of such compounds is fully describedin the aforementioned patents and publications, which are incorporatedherein by reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the compounds of the present invention is selectedfrom:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)-phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

A compound of the instant invention may also be administered with anagent useful in the treatment of anemia. Such an anemia treatment agentis, for example, a continuous eythropoiesis receptor activator (such asepoetin alfa).

A compound of the instant invention may also be administered with anagent useful in the treatment of neutropenia. Such a neutropeniatreatment agent is, for example, a hematopoietic growth factor whichregulates the production and function of neutrophils such as a humangranulocyte colony stimulating factor, (G-CSF). Examples of a G-CSFinclude filgrastim and PEG-filgrastim.

A compound of the instant invention may also be administered with animmunologic-enhancing drug, such as levamisole, isoprinosine andZadaxin.

A compound of the instant invention may also be useful for treating orpreventing liver disease or cancer in combination with other siNAtherapeutics.

The compounds of the instant invention may also be administered incombination with γ-secretase inhibitors and/or inhibitors of NOTCHsignaling. Such inhibitors include compounds described in WO 01/90084,WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370,WO 2005/030731, WO 2005/014553, U.S. Ser. No. 10/957,251, WO2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137,WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO02/47671 (including LY-450139).

A compound of the instant invention may also be useful for treating orpreventing cancer in combination with PARP inhibitors.

A compound of the instant invention may also be useful for treatingcancer in combination with the following therapeutic agents: abarelix(Plenaxis Depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®);Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol(Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole(Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®);azacitidine (Vidaza®); bendamustine hydrochloride (Treanda®);bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel(Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefeldinA; busulfan intravenous (Busulfex®); busulfan oral (Myleran®);calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin(Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®);carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib(Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin(Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®);cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (CytoxanInjection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection(Fragmin®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix(Firmagon®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®);dexrazoxane hydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel(Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®,Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal(Doxil®); dromostanolone propionate (Dromostanolone®); dromostanolonepropionate (Masterone Injection®); eculizumab injection (Soliris®);Elliott's B Solution (Elliott's B Solution®); eltrombopag (Promacta®);epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®);estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate(Etopophos®); etoposide, VP-16 (Vepesid®); everolimus tablets(Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®);Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine(Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®);gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumabozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelinacetate (Zoladex®); histrelin acetate (Histrelin Implant®); hydroxyurea(Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®);ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a(Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 injection(AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinibtablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femora®);leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®);levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine,nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan,L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®);mesna (Mesnex Tabs®); methotrexate (Methotrexate®); methoxsalen(Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane(Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib(Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin(Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel(Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin(Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanibtablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®);pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium(Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor(Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium(Photofrin®); pralatrexate injection (Folotyn®); procarbazine(Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®);raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin(Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim(Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinibmaleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); ternsirolimus (Torisel®); teniposide, VM-26(Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®);thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene(Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab(Bexxar®); trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin,ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil MustardCapsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine(Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin;and zoledronate (Zometa®).

The combinations referred to above can conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

The individual compounds of such combinations can be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. In one embodiment, the individual compounds will beadministered simultaneously in a combined pharmaceutical formulation.

Thus, the described molecules could be used in combination with one ormore known compounds, treatments, or procedures to prevent or treatdiseases, disorders, conditions, and traits described herein in asubject or organism as are known in the art, such as other HBVinhibitors.

3. Therapeutic Applications

The present body of knowledge in HBV research indicates the need formethods that can regulate HBV expression for therapeutic use.

Thus, one aspect of the invention comprises a method of treating asubject including, but not limited to, a human suffering HBV infectionor a condition which is mediated by the action of HBV gene expression,which method comprises administering to said subject an effective amountof a double-stranded siNA molecule of the invention. In one embodimentof this aspect, the siNA molecules comprises at least a 15 nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ ID NO: 2, SEQ ID NO: 453,SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, or SEQ ID NO:455; or formula(A).

In some embodiments of this aspect, the condition is cancer. Thus, incertain embodiments the molecules and compositions of the instantinvention are useful in a method for treating cancer, and in particular,in treating hepatocellular carcinoma (HCC).

In some embodiments, the condition is liver disease. Thus, in certainembodiments the molecules and compositions of the instant invention areuseful in a method for treating liver disease, and, in particular, intreating cirrhosis of the liver.

In certain embodiments, the administration of the siNA molecule is vialocal administration or systemic administration. In other embodiments,the invention features contacting the subject or organism with an siNAmolecule of the invention via local administration to relevant tissuesor cells, such as lung cells and tissues, such as via pulmonarydelivery. In yet other embodiments, the invention features contactingthe subject or organism with an siNA molecule of the invention viasystemic administration (such as via intravenous or subcutaneousadministration of siNA) to relevant tissues or cells, such as liver orcancerous tissues or cells in a subject or organism.

siNA molecules of the invention are also used as reagents in ex vivoapplications. For example, siNA reagents are introduced into tissue orcells that are transplanted into a subject for therapeutic effect. Thecells and/or tissue can be derived from an organism or subject thatlater receives the explant, or can be derived from another organism orsubject prior to transplantation. The siNA molecules can be used tomodulate the expression of one or more genes in the cells or tissue,such that the cells or tissue obtain a desired phenotype or are able toperform a function when transplanted in vivo. In one embodiment, certainHBV target cells from a patient are extracted. These extracted cells arecontacted with HBV siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g., using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients.

For therapeutic applications, a pharmaceutically effective dose of thesiNA molecules or pharmaceutical compositions of the invention isadministered to the subject. A pharmaceutically effective dose is thatdose required to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) a disease state.One skilled in the art can readily determine a therapeutically effectivedose of the siNA of the invention to be administered to a given subject,by taking into account factors, such as the size and weight of thesubject, the extent of the disease progression or penetration, the age,health, and sex of the subject, the route of administration, and whetherthe administration is regional or systemic. Generally, an amount between0.1 μg/kg and 100 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the negatively charged polymer.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. The siNA molecules of theinvention can be administered in a single dose or in multiple doses.

siNA molecules of the instant invention can be administered oncemonthly, once weekly, once daily (QD), or divided into multiple monthly,weekly, or daily doses, such as, for example, but not limitation, twicedaily (BID), three times daily (TID), once every two weeks. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues.

In addition, the administration can be continuous, i.e., every day, orintermittently. For example, intermittent administration of a compoundof the instant invention may be administration one to six days per weekor it may mean administration in cycles (e.g. daily administration fortwo to eight consecutive weeks, then a rest period with noadministration for up to one week) or it may mean administration onalternate days.

G. Administration

Compositions or formulations can be administered in a variety of ways.Non-limiting examples of administration methods of the invention includeoral, buccal, sublingual, parenteral (i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly),local rectal administration or other local administration. In oneembodiment, the composition of the invention can be administered byinsufflation and inhalation. Administration can be accomplished viasingle or divided doses. In some embodiments, the pharmaceuticalcompositions are administered intravenously or intraperitoneally by abolus injection (see, e.g., U.S. Pat. No. 5,286,634). Lipid nucleic acidparticles can be administered by direct injection at the site of diseaseor by injection at a site distal from the site of disease (see, e.g.,Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York.pp. 70-71(1994)). In one embodiment, the siNA molecules of the inventionand formulations or compositions thereof are administered to a cell,subject, or organism as is described herein and as is generally known inthe art.

1. In Vivo Administration

In any of the methods of treatment of the invention, the siNA can beadministered to the subject systemically as described herein orotherwise known in the art, either alone as a monotherapy or incombination with additional therapies described herein or as are knownin the art. Systemic administration can include, for example, pulmonary(inhalation, nebulization etc.) intravenous, subcutaneous,intramuscular, catheterization, nasopharangeal, transdermal, ororal/gastrointestinal administration as is generally known in the art.

In any of the methods of treatment or prevention of the invention, thesiNA can be administered to the subject locally or to local tissues asdescribed herein or otherwise known in the art, either alone as amonotherapy or in combination with additional therapies as are known inthe art. Local administration can include, for example, inhalation,nebulization, catheterization, implantation, direct injection,dermal/transdermal application, patches, stenting, ear/eye drops, orportal vein administration to relevant tissues, or any other localadministration technique, method or procedure, as is generally known inthe art.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the liver as is generallyknown in the art (see for example Wen et al., 2004, World JGastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14;Liu et al., 2003, gene Ther., 10, 180-7; Hong et al., 2003, J PharmPharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7;and Matsuno et al., 2003, gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliverthe siNA molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles as is generally known in the art(see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnieret al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs,16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; andPreat and Dujardin, 2001, STP PharmaSciences, 11, 57-68). In oneembodiment, the siNA molecules of the invention and formulations orcompositions thereof are administered directly or topically using ahydroalcoholic gel formulation comprising an alcohol (e.g., ethanol orisopropanol), water, and optionally including additional agents suchisopropyl myristate and carbomer 980. In other embodiments, the siNA areformulated to be administered topically to the nasal cavity. Topicalpreparations can be administered by one or more applications per day tothe affected area; over skin areas occlusive dressings canadvantageously be used. Continuous or prolonged delivery can be achievedby an adhesive reservoir system.

In one embodiment, an siNA molecule of the invention is administerediontophoretically, for example to a particular organ or compartment(e.g., the eye, back of the eye, heart, liver, kidney, bladder,prostate, tumor, CNS etc.). Non-limiting examples of iontophoreticdelivery are described in, for example, WO 03/043689 and WO 03/030989,which are incorporated by reference in their entireties herein.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the lung as is describedherein and as is generally known in the art. In another embodiment, thesiNA molecules of the invention and formulations or compositions thereofare administered to lung tissues and cells as is described in U.S.Patent Publication Nos. 2006/0062758; 2006/0014289; and 2004/0077540.

2. Aerosols and Delivery Devices

a. Aerosol Formulations

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In one embodiment, the siNA molecules of the invention and formulationsthereof are administered via pulmonary delivery, such as by inhalationof an aerosol or spray dried formulation administered by an inhalationdevice or nebulizer, providing rapid local uptake of the nucleic acidmolecules into relevant pulmonary tissues. Solid particulatecompositions containing respirable dry particles of micronized nucleicacid compositions can be prepared by grinding dried or lyophilizednucleic acid compositions, and then passing the micronized compositionthrough, for example, a 400 mesh screen to break up or separate outlarge agglomerates. A solid particulate composition comprising the siNAcompositions of the invention can optionally contain a dispersant whichserves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Spray compositions comprising siNA molecules or compositions of theinvention can, for example, be formulated as aqueous solutions orsuspensions or as aerosols delivered from pressurized packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.In one embodiment, aerosol compositions of the invention suitable forinhalation can be either a suspension or a solution and generallycontain an siNA molecule comprising at least a 15 nucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 452, SEQ ID NO: 2, SEQ ID NO: 453, SEQ ID NO:3, SEQ ID NO: 454, SEQ ID NO: 4, or SEQ ID NO:455; or formula (A), and asuitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition can optionally contain additional formulation excipientswell known in the art such as surfactants. Non-limiting examples includeoleic acid, lecithin or an oligolactic acid or derivative such as thosedescribed in WO94/21229 and WO98/34596 and co-solvents for exampleethanol. In one embodiment a pharmaceutical aerosol formulation of theinvention comprising a compound of the invention and a fluorocarbon orhydrogen-containing chlorofluorocarbon or mixtures thereof aspropellant, optionally in combination with a surfactant and/or aco-solvent.

The aerosol formulations of the invention can be buffered by theaddition of suitable buffering agents.

Aerosol formulations can include optional additives includingpreservatives if the formulation is not prepared sterile. Non-limitingexamples include, methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. In one embodiment, fluorocarbon or perfluorocarbon carriersare used to reduce degradation and provide safer biocompatiblenon-liquid particulate suspension compositions of the invention (e.g.,siNA and/or LNP formulations thereof). In another embodiment, a devicecomprising a nebulizer delivers a composition of the invention (e.g.,siNA and/or LNP formulations thereof) comprising fluorochemicals thatare bacteriostatic thereby decreasing the potential for microbial growthin compatible devices.

Capsules and cartridges comprising the composition of the invention foruse in an inhaler or insufflator, of for example gelatine, can beformulated containing a powder mix for inhalation of a compound of theinvention and a suitable powder base such as lactose or starch. In oneembodiment, each capsule or cartridge contains an siNA moleculecomprising at least a 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:452, SEQ ID NO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO: 454, SEQ IDNO: 4, or SEQ ID NO:455; or formula (A), and one or more excipients. Inanother embodiment, the compound of the invention can be presentedwithout excipients such as lactose

The aerosol compositions of the present invention can be administeredinto the respiratory system as a formulation including particles ofrespirable size, e.g. particles of a size sufficiently small to passthrough the nose, mouth and larynx upon inhalation and through thebronchi and alveoli of the lungs. In general, respirable particles rangefrom about 0.5 to 10 microns in size. In one embodiment, the particulaterange can be from 1 to 5 microns. In another embodiment, the particulaterange can be from 2 to 3 microns. Particles of non-respirable size whichare included in the aerosol tend to deposit in the throat and beswallowed, and the quantity of non-respirable particles in the aerosolis thus minimized. For nasal administration, a particle size in therange of 10-500 um is preferred to ensure retention in the nasal cavity.

In some embodiments, an siNA composition of the invention isadministered topically to the nose for example, for the treatment ofrhinitis, via pressurized aerosol formulations, aqueous formulationsadministered to the nose by pressurized pump or by nebulization.Suitable formulations contain water as the diluent or carrier for thispurpose. In certain embodiments, the aqueous formulations foradministration of the composition of the invention to the lung or nosecan be provided with conventional excipients such as buffering agents,tonicity modifying agents and the like.

b. Devices

The siNA molecules of the invention can be formulated and delivered asparticles and/or aerosols as discussed above and dispensed from variousaerosolization devices known by those of skill in the art.

Aerosols of liquid or non-liquid particles comprising an siNA moleculeor formulation of the invention can be produced by any suitable means,such as with a device comprising a nebulizer (see for example U.S. Pat.No. 4,501,729) such as ultrasonic or air jet nebulizers.

Solid particle aerosols comprising an siNA molecule or formulation ofthe invention and surfactant can be produced with any solid particulateaerosol generator. One type of solid particle aerosol generator usedwith the siNA molecules of the invention is an insufflator. A secondtype of illustrative aerosol generator comprises a metered dose inhaler(“MDI”). MDIs containing siNA molecules or formulations taught hereincan be prepared by methods of the art (for example, see Byron, above andWO96/32099).

The siNA molecules can also be formulated as a fluid formulation fordelivery from a fluid dispenser, such as those described and illustratedin WO05/044354.

In certain embodiments of the invention, nebulizer devices are used inapplications for conscious, spontaneously breathing subjects, and forcontrolled ventilated subjects of all ages. The nebulizer devices can beused for targeted topical and systemic drug delivery to the lung. In oneembodiment, a device comprising a nebulizer is used to deliver an siNAmolecule or formulation of the invention locally to lung or pulmonarytissues. In another embodiment, a device comprising a nebulizer is usedto deliver a an siNA molecule or formulation of the inventionsystemically.

H. Other Applications/Uses of siNA Molecules of the Invention

The siNA molecules of the invention can also be used for diagnosticapplications, research applications, and/or manufacture of medicants.

In one aspect, the invention features a method for diagnosing a disease,trait, or condition in a subject comprising administering to the subjecta composition of the invention under conditions suitable for thediagnosis of the disease, trait, or condition in the subject.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of HBV proteins arising fromhaplotype polymorphisms that are associated with a trait, disease orcondition in a subject or organism. Analysis of HBV genes, or HBVprotein or RNA levels can be used to identify subjects with suchpolymorphisms or those subjects who are at risk of developing traits,conditions, or diseases described herein. These subjects are amenable totreatment, for example, treatment with siNA molecules of the inventionand any other composition useful in treating diseases related to targetgene expression. As such, analysis of HBV protein or RNA levels can beused to determine treatment type and the course of therapy in treating asubject. Monitoring of HBV protein (e.g., hepatitis B core antigen, orHbcAg), HBV surface antigen (e.g., HBsAg) or RNA levels can be used topredict treatment outcome and to determine the efficacy of compounds andcompositions that modulate the level and/or activity of certain HBVproteins associated with a trait, disorder, condition, or disease.

In another embodiment, the invention comprises use of a double-strandednucleic acid according to the invention for use in the manufacture of amedicament. In an embodiment, the medicament is for use in treating HBVinfection or a condition that is mediated by the action of HBV. In oneembodiment, the medicant is for use in treating HBV infection. In someembodiments, the medicament is for use in the treatment of cancer. In aparticular embodiment, the compounds of the instant invention are usefulfor treating hepatocellular carcinoma. In one embodiment, the medicamentis for use in the treatment of liver disease. In a particularembodiment, the compounds of the instant invention are useful fortreating cirrhosis of the liver.

In certain embodiments, siNAs wherein at least one strand comprises atleast a 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ IDNO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, orSEQ ID NO:455; or formula (A), are for use in a method for treating HBVinfection.

In certain embodiments, siNAs wherein at least one strand comprises atleast a 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ IDNO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, orSEQ ID NO:455; or formula (A), are for use in a method for treatingcancer.

In certain embodiments, siNAs wherein at least one strand comprises atleast a 15 nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 452, SEQ IDNO: 2, SEQ ID NO: 453, SEQ ID NO: 3, SEQ ID NO: 454, SEQ ID NO: 4, orSEQ ID NO:455; or formula (A), are for use in a method for treatingliver disease.

I. Examples

The invention will now be illustrated with the following non-limitingexamples. Those of skill in the art will readily recognize a variety ofnon-critical parameters which can be changed or modified to yieldessentially the same results.

Example 1: Design, Synthesis, and Identification of siNAs Active AgainstHBV

HBV siNA Synthesis

A series of siNA molecules were designed, synthesized and evaluated forefficacy against HBV gene expression. HBV sequences were designed andselected by selecting all possible 19 nucleotide sequences from HBVgenome serotype adw2 (Accession Number X02763) and then comparing thesesequences to the genome sequences of 22 other HBV subtypes shown inTable 7. 19 nucleotide sequences having perfect matches to all 22genomes, or perfect matches in 21 of the 22 genomes and 1 or 2mismatches to the remaining genome, were retained. These sequences weresubjected to standard filters (e.g., off-target to human genome, runs ofsame base, matches to human miRNA seed sequences) (See also U.S.Application No. 60/182,605 for sequence design methods and standardfilters). The remaining candidates were sorted by a proprietarysilencing prediction score and certain sequences were selected.

The primary criteria for design for the HBV sequences for human siNAswere (i) homology between multiple HBV genotypes and serotypes and (ii)high efficacy scores as determined by a proprietary algorithm. Thetarget sequences of the siNAs that were selected are set forth in Table1a (target sequences). The sense and antisense strands of the siNAsequences corresponding to the target sequences in Table 1a are setforth in Table 1b. Various chemically modified siNAs that weresynthesized are set forth in Table 1c.

TABLE 1a HBV Target Sequences, noting target sites. Target SequenceTarget Site SEQ ID NO: UCGUGGUGGACUUCUCUCA 1663 1 GUGGUGGACUUCUCUCAAU1665 2 GCCGAUCCAUACUGCGGAA 2669 3 CCGAUCCAUACUGCGGAAC 2670 4CAUCCUGCUGCUAUGCCUC 1818 5 UGCUGCUAUGCCUCAUCUU 1823 6GGUGGACUUCUCUCAAUUU 1667 7 UGGUGGACUUCUCUCAAUU 1666 8UAGACUCGUGGUGGACUUC 1658 9 UCCUCUGCCGAUCCAUACU 2663 10UGCCGAUCCAUACUGCGGA 2668 11 UGGAUGUGUCUGCGGCGUU 1783 12CGAUCCAUACUGCGGAACU 2671 13 CGCACCUCUCUUUACGCGG 2934 14CUGCCGAUCCAUACUGCGG 2667 15 CGUGGUGGACUUCUCUCAA 1664 16CUGCUGCUAUGCCUCAUCU 1822 17 CCUGCUGCUAUGCCUCAUC 1821 18CUAGACUCGUGGUGGACUU 1657 19 UCCUGCUGCUAUGCCUCAU 1820 20GACUCGUGGUGGACUUCUC 1660 21 AUCCAUACUGCGGAACUCC 2673 22CUCUGCCGAUCCAUACUGC 2665 23 GAUCCAUACUGCGGAACUC 2672 24GAAGAACUCCCUCGCCUCG 567 25 AAGCCUCCAAGCUGUGCCU 54 26 AGAAGAACUCCCUCGCCUC566 27 GGAGUGUGGAUUCGCACUC 455 28 CCUCUGCCGAUCCAUACUG 2664 29CAAGCCUCCAAGCUGUGCC 53 30 UCCAUACUGCGGAACUCCU 2674 31CAGAGUCUAGACUCGUGGU 1651 32 AAGAAGAACUCCCUCGCCU 565 33GAGUGUGGAUUCGCACUCC 456 34 UCUAGACUCGUGGUGGACU 1656 35GCUGCUAUGCCUCAUCUUC 1824 36 AGUCUAGACUCGUGGUGGA 1654 37CUCCUCUGCCGAUCCAUAC 2662 38 UGGCUCAGUUUACUAGUGC 2077 39GUCUAGACUCGUGGUGGAC 1655 40 UUCAAGCCUCCAAGCUGUG 51 41CUAUGGGAGUGGGCCUCAG 2047 42 CUCGUGGUGGACUUCUCUC 1662 43CCUAUGGGAGUGGGCCUCA 2046 44 AAGAACUCCCUCGCCUCGC 568 45UCUGCCGAUCCAUACUGCG 2666 46 AGAGUCUAGACUCGUGGUG 1652 47GAAGAAGAACUCCCUCGCC 564 48 UCAAGCCUCCAAGCUGUGC 52 49 AGCCUCCAAGCUGUGCCUU55 50 AGACUCGUGGUGGACUUCU 1659 51

TABLE 1b Various HBV siNA sense and antisense sequences correspondingto the identified target sequences in Table 1a. Target SEQ ID SEQ IDSite NO: Sense Sequence Antisense Sequence NO: 1663 1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGA 452 1665 2 GUGGUGGACUUCUCUCAAUAUUGAGAGAAGUCCACCAC 453 2669 3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGC454 2670 4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGG 455 1818 5CAUCCUGCUGCUAUGCCUC GAGGCAUAGCAGCAGGAUG 456 1823 6 UGCUGCUAUGCCUCAUCUUAAGAUGAGGCAUAGCAGCA 457 1667 7 GGUGGACUUCUCUCAAUUU AAAUUGAGAGAAGUCCACC458 1666 8 UGGUGGACUUCUCUCAAUU AAUUGAGAGAAGUCCACCA 459 1658 9UAGACUCGUGGUGGACUUC GAAGUCCACCACGAGUCUA 460 2663 10 UCCUCUGCCGAUCCAUACUAGUAUGGAUCGGCAGAGGA 461 2668 11 UGCCGAUCCAUACUGCGGA UCCGCAGUAUGGAUCGGCA462 1783 12 UGGAUGUGUCUGCGGCGUU AACGCCGCAGACACAUCCA 463 2671 13CGAUCCAUACUGCGGAACU AGUUCCGCAGUAUGGAUCG 464 2934 14 CGCACCUCUCUUUACGCGGCCGCGUAAAGAGAGGUGCG 465 2667 15 CUGCCGAUCCAUACUGCGG CCGCAGUAUGGAUCGGCAG466 1664 16 CGUGGUGGACUUCUCUCAA UUGAGAGAAGUCCACCACG 467 1822 17CUGCUGCUAUGCCUCAUCU AGAUGAGGCAUAGCAGCAG 468 1821 18 CCUGCUGCUAUGCCUCAUCGAUGAGGCAUAGCAGCAGG 469 1657 19 CUAGACUCGUGGUGGACUU AAGUCCACCACGAGUCUAG470 1820 20 UCCUGCUGCUAUGCCUCAU AUGAGGCAUAGCAGCAGGA 471 1660 21GACUCGUGGUGGACUUCUC GAGAAGUCCACCACGAGUC 472 2673 22 AUCCAUACUGCGGAACUCCGGAGUUCCGCAGUAUGGAU 473 2665 23 CUCUGCCGAUCCAUACUGC GCAGUAUGGAUCGGCAGAG474 2672 24 GAUCCAUACUGCGGAACUC GAGUUCCGCAGUAUGGAUC 475 567 25GAAGAACUCCCUCGCCUCG CGAGGCGAGGGAGUUCUUC 476 54 26 AAGCCUCCAAGCUGUGCCUAGGCACAGCUUGGAGGCUU 477 566 27 AGAAGAACUCCCUCGCCUC GAGGCGAGGGAGUUCUUCU478 455 28 GGAGUGUGGAUUCGCACUC GAGUGCGAAUCCACACUCC 479 2664 29CCUCUGCCGAUCCAUACUG CAGUAUGGAUCGGCAGAGG 480 53 30 CAAGCCUCCAAGCUGUGCCGGCACAGCUUGGAGGCUUG 481 2674 31 UCCAUACUGCGGAACUCCU AGGAGUUCCGCAGUAUGGA482 1651 32 CAGAGUCUAGACUCGUGGU ACCACGAGUCUAGACUCUG 483 565 33AAGAAGAACUCCCUCGCCU AGGCGAGGGAGUUCUUCUU 484 456 34 GAGUGUGGAUUCGCACUCCGGAGUGCGAAUCCACACUC 485 1656 35 UCUAGACUCGUGGUGGACU AGUCCACCACGAGUCUAGA486 1824 36 GCUGCUAUGCCUCAUCUUC GAAGAUGAGGCAUAGCAGC 487 1654 37AGUCUAGACUCGUGGUGGA UCCACCACGAGUCUAGACU 488 2662 38 CUCCUCUGCCGAUCCAUACGUAUGGAUCGGCAGAGGAG 489 2077 39 UGGCUCAGUUUACUAGUGC GCACUAGUAAACUGAGCCA490 1655 40 GUCUAGACUCGUGGUGGAC GUCCACCACGAGUCUAGAC 491 51 41UUCAAGCCUCCAAGCUGUG CACAGCUUGGAGGCUUGAA 492 2047 42 CUAUGGGAGUGGGCCUCAGCUGAGGCCCACUCCCAUAG 493 1662 43 CUCGUGGUGGACUUCUCUC GAGAGAAGUCCACCACGAG494 2046 44 CCUAUGGGAGUGGGCCUCA UGAGGCCCACUCCCAUAGG 495 568 45AAGAACUCCCUCGCCUCGC GCGAGGCGAGGGAGUUCUU 496 2666 46 UCUGCCGAUCCAUACUGCGCGCAGUAUGGAUCGGCAGA 497 1652 47 AGAGUCUAGACUCGUGGUG CACCACGAGUCUAGACUCU498 564 48 GAAGAAGAACUCCCUCGCC GGCGAGGGAGUUCUUCUUC 499 52 49UCAAGCCUCCAAGCUGUGC GCACAGCUUGGAGGCUUGA 500 55 50 AGCCUCCAAGCUGUGCCUUAAGGCACAGCUUGGAGGCU 501 1659 51 AGACUCGUGGUGGACUUCU AGAAGUCCACCACGAGUCU502

For each oligonucleotide of a target sequence, the two individual,complementary strands of the siNA were synthesized separately usingsolid phase synthesis, then purified separately by reversed phase solidphase extraction (SPE). The complementary strands were annealed to formthe double strand (duplex) and delivered in the desired concentrationand buffer of choice.

Briefly, the single strand oligonucleotides were synthesized usingphosphoramidite chemistry on an automated solid-phase synthesizer, usingprocedures as are generally known in the art (see for example U.S.application Ser. No. 12/064,014). A synthesis column was packed withsolid support derivatized with the first nucleoside residue (natural orchemically modified). Synthesis was initiated by detritylation of theacid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. Asuitably protected phosphoramidite and a suitable activator inacetonitrile were delivered simultaneously to the synthesis columnresulting in coupling of the amidite to the 5′-hydroxyl. The column wasthen washed with a solvent, such as acetonitrile. An oxidizing solution,such as an iodine solution was pumped through the column to oxidize thephosphite triester linkage P(III) to its phosphotriester P(V) analog.Unreacted 5′-hydroxyl groups were capped using reagents such as aceticanhydride in the presence of 2,6-lutidine and N-methylimidazole. Theelongation cycle was resumed with the detritylation step for the nextphosphoramidite incorporation. This process was repeated until thedesired sequence was synthesized. The synthesis concluded with the final5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).

Upon completion of the synthesis, the solid-support and associatedoligonucleotide were dried under argon pressure or vacuum. Aqueous basewas added and the mixture was heated to effect cleavage of the succinyllinkage, removal of the cyanoethyl phosphate protecting group, anddeprotection of the exocyclic amine protection.

The following process was performed on single strands that do notcontain ribonucleotides. After treating the solid support with theaqueous base, the mixture was filtered to separate the solid supportfrom the deprotected crude synthesis material. The solid support wasthen rinsed with water, which is combined with the filtrate. Theresultant basic solution allows for retention of the5′-O-dimethoxytrityl group to remain on the 5′ terminal position(trityl-on).

For single strands that contain ribonucleotides, the following processwas performed. After treating the solid support with the aqueous base,the mixture was filtered to separate the solid support from thedeprotected crude synthesis material. The solid support was then rinsedwith dimethylsulfoxide (DMSO), which was combined with the filtrate.Fluoride reagent, such as triethylamine trihydrofluoride, was added tothe mixture, and the solution was heated. The reaction was quenched withsuitable buffer to provide a solution of crude single strand with the5′-O-dimethoxytrityl group on the final 5′ terminal position.

The trityl-on solution of each crude single strand was purified usingchromatographic purification, such as SPE RPC purification. Thehydrophobic nature of the trityl group permits stronger retention of thedesired full-length oligo than the non-tritylated truncated failuresequences. The failure sequences were selectively washed from the resinwith a suitable solvent, such as low percent acetonitrile. Retainedoligonucleotides were then detritylated on-column with trifluoroaceticacid to remove the acid-labile trityl group. Residual acid was washedfrom the column, a salt exchange was performed, and a final desalting ofthe material commenced. The full-length oligo was recovered in apurified form with an aqueous-organic solvent. The final product wasthen analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UVA₂₆₀). The oligos were dried via lyophilization or vacuum condensation.

Annealing: Based on the analysis of the product, the dried oligos weredissolved in appropriate buffers followed by mixing equal molar amounts(calculated using the theoretical extinction coefficient) of the senseand antisense oligonucleotide strands. The solution was then analyzedfor purity of duplex by chromatographic methods and desired finalconcentration. If the analysis indicated an excess of either strand,then the additional non-excess strand was titrated until duplexing wascomplete. When analysis indicated that the target product purity hasbeen achieved the material was delivered and ready for use.

Below is a table showing various modified siNAs synthesized using thisprotocol or that can be synthesized using this protocol or using methodsknown in the art.

TABLE 1cHBV siNA Strands Synthesized (Antisense sequences are readily identifiedas having complete or partial complementarity to the target sequence shown).SEQ SEQ Target ID ID siNA duplex ID Site NO: Target SequenceModified Sequence NO: R-008351268-000C 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCATT B  52 R-008351268-000C 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUU  53 R-008351278-000V 1665  2GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuTT B  54 R-008351278-000V 1665 2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUU  55 R-008351342-000A 2669 3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAATT B  56 R-008351342-000A2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUU  57 R-008351172-000H1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUTT B  58R-008351172-000H 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUU  59R-008351362-000K 2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcTT B 60 R-008351362-000K 2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUU 61 R-008351389-000F 1818  5 CAUCCUGCUGCUAUGCCUCB CAUCCUGCUGCUAUGCCUCTT B  62 R-008351389-000F 1818  5CAUCCUGCUGCUAUGCCUC GAGGCAUAGCAGCAGGAUGUU  63 R-008351306-000R 1823  6UGCUGCUAUGCCUCAUCUU B UGCUGCUAUGCCUCAUCUUTT B  64 R-008351306-000R 1823 6 UGCUGCUAUGCCUCAUCUU AAGAUGAGGCAUAGCAGCAUU  65 R-008351372-000C 1667 7 GGUGGACUUCUCUCAAUUU B GGuGGAcuucucucAAuuuTT B  66 R-008351372-000C1667  7 GGUGGACUUCUCUCAAUUU AAAuuGAGAGAAGuccAccUU  67 R-008351317-000S1666  8 UGGUGGACUUCUCUCAAUU B uGGuGGAcuucucucAAuuTT B  68R-008351317-000S 1666  8 UGGUGGACUUCUCUCAAUU AAUuGAGAGAAGuccAccAUU  69R-008351210-000W 1658  9 UAGACUCGUGGUGGACUUC B UAGACUCGUGGUGGACUUCTT B 70 R-008351210-000W 1658  9 UAGACUCGUGGUGGACUUC GAAGUCCACCACGAGUCUAUU 71 R-008351329-000B 2663 10 UCCUCUGCCGAUCCAUACUB UCCUCUGCCGAUCCAUACUTT B  72 R-008351329-000B 2663 10UCCUCUGCCGAUCCAUACU AGUAUGGAUCGGCAGAGGAUU  73 R-008351350-000A 1658  9UAGACUCGUGGUGGACUUC B uAGAcucGuGGuGGAcuucTT B  74 R-008351350-000A 1658 9 UAGACUCGUGGUGGACUUC GAAGuccAccAcGAGucuAUU  75 R-008351303-000P 266811 UGCCGAUCCAUACUGCGGA B UGCCGAUCCAUACUGCGGATT B  76 R-008351303-000P2668 11 UGCCGAUCCAUACUGCGGA UCCGCAGUAUGGAUCGGCAUU  77 R-008351229-000S1783 12 UGGAUGUGUCUGCGGCGUU B UGGAUGUGUCUGCGGCGUUTT B  78R-008351229-000S 1783 12 UGGAUGUGUCUGCGGCGUU AACGCCGCAGACACAUCCAUU  79R-008351386-000E 2671 13 CGAUCCAUACUGCGGAACU B CGAUCCAUACUGCGGAACUTT B 80 R-008351386-000E 2671 13 CGAUCCAUACUGCGGAACU AGUUCCGCAGUAUGGAUCGUU 81 R-008351300-000N 2934 14 CGCACCUCUCUUUACGCGGB CGCACCUCUCUUUACGCGGTT B  82 R-008351300-000N 2934 14CGCACCUCUCUUUACGCGG CCGCGUAAAGAGAGGUGCGUU  83 R-008351263-000J 2667 15CUGCCGAUCCAUACUGCGG B CUGCCGAUCCAUACUGCGGTT B  84 R-008351263-000J 266715 CUGCCGAUCCAUACUGCGG CCGCAGUAUGGAUCGGCAGUU  85 R-008351190-000A 178312 UGGAUGUGUCUGCGGCGUU B uGGAuGuGucuGcGGcGuuTT B  86 R-008351190-000A1783 12 UGGAUGUGUCUGCGGCGUU AACGccGcAGAcAcAuccAUU  87 R-008351326-000A1666  8 UGGUGGACUUCUCUCAAUU B UGGUGGACUUCUCUCAAUUTT B  88R-008351326-000A 1666  8 UGGUGGACUUCUCUCAAUU AAUUGAGAGAAGUCCACCAUU  89R-008351260-000H 1664 16 CGUGGUGGACUUCUCUCAA B CGUGGUGGACUUCUCUCAATT B 90 R-008351260-000H 1664 16 CGUGGUGGACUUCUCUCAA UUGAGAGAAGUCCACCACGUU 91 R-008351195-000U 1822 17 CUGCUGCUAUGCCUCAUCUB CUGCUGCUAUGCCUCAUCUTT B  92 R-008351195-000U 1822 17CUGCUGCUAUGCCUCAUCU AGAUGAGGCAUAGCAGCAGUU  93 R-008351323-000Z 1821 18CCUGCUGCUAUGCCUCAUC B CCUGCUGCUAUGCCUCAUCTT B  94 R-008351323-000Z 182118 CCUGCUGCUAUGCCUCAUC GAUGAGGCAUAGCAGCAGGUU  95 R-008351367-000D 1667 7 GGUGGACUUCUCUCAAUUU B GGUGGACUUCUCUCAAUUUTT B  96 R-008351367-000D1667  7 GGUGGACUUCUCUCAAUUU AAAUUGAGAGAAGUCCACCUU  97 R-008351251-000Z1657 19 CUAGACUCGUGGUGGACUU B cuAGAcucGuGGuGGAcuuTT B  98R-008351251-000Z 1657 19 CUAGACUCGUGGUGGACUU AAGuccAccAcGAGucuAGUU  99R-008351240-000Y 1820 20 UCCUGCUGCUAUGCCUCAU B UCCUGCUGCUAUGCCUCAUTT B100 R-008351240-000Y 1820 20 UCCUGCUGCUAUGCCUCAU AUGAGGCAUAGCAGCAGGAUU101 R-008351178-000K 2668 11 UGCCGAUCCAUACUGCGGAB uGccGAuccAuAcuGcGGATT B 102 R-008351178-000K 2668 11UGCCGAUCCAUACUGCGGA UCCGcAGuAuGGAucGGcAUU 103 R-008351353-000B 1657 19CUAGACUCGUGGUGGACUU B CUAGACUCGUGGUGGACUUTT B 104 R-008351353-000B 165719 CUAGACUCGUGGUGGACUU AAGUCCACCACGAGUCUAGUU 105 R-008351297-000W 166021 GACUCGUGGUGGACUUCUC B GACUCGUGGUGGACUUCUCTT B 106 R-008351297-000W1660 21 GACUCGUGGUGGACUUCUC GAGAAGUCCACCACGAGUCUU 107 R-008351175-000J2673 22 AUCCAUACUGCGGAACUCC B AuccAuAcuGcGGAAcuccTT B 108R-008351175-000J 2673 22 AUCCAUACUGCGGAACUCC GGAGuuccGcAGuAuGGAuUU 109R-008351181-000S 2665 23 CUCUGCCGAUCCAUACUGC B cucuGccGAuccAuAcuGcTT B110 R-008351181-000S 2665 23 CUCUGCCGAUCCAUACUGC GCAGuAuGGAucGGcAGAGUU111 R-008351184-000T 2672 24 GAUCCAUACUGCGGAACUCB GAUCCAUACUGCGGAACUCTT B 112 R-008351184-000T 2672 24GAUCCAUACUGCGGAACUC GAGUUCCGCAGUAUGGAUCUU 113 R-008351187-000U  567 25GAAGAACUCCCUCGCCUCG B GAAGAACUCCCUCGCCUCGTT B 114 R-008351187-000U  56725 GAAGAACUCCCUCGCCUCG CGAGGCGAGGGAGUUCUUCUU 115 R-008351192-000T   5426 AAGCCUCCAAGCUGUGCCU B AAGccuccAAGcuGuGccuTT B 116 R-008351192-000T  54 26 AAGCCUCCAAGCUGUGCCU AGGcAcAGcuuGGAGGcuuUU 117 R-008351198-000V 566 27 AGAAGAACUCCCUCGCCUC B AGAAGAACUCCCUCGCCUCTT B 118R-008351198-000V  566 27 AGAAGAACUCCCUCGCCUC GAGGCGAGGGAGUUCUUCUUU 119R-008351201-000M 2671 13 CGAUCCAUACUGCGGAACU B cGAuccAuAcuGcGGAAcuTT B120 R-008351201-000M 2671 13 CGAUCCAUACUGCGGAACU AGUuccGcAGuAuGGAucGUU121 R-008351204-000N  455 28 GGAGUGUGGAUUCGCACUCB GGAGUGUGGAUUCGCACUCTT B 122 R-008351204-000N  455 28GGAGUGUGGAUUCGCACUC GAGUGCGAAUCCACACUCCUU 123 R-008351207-000P 2664 29CCUCUGCCGAUCCAUACUG B ccucuGccGAuccAuAcuGTT B 124 R-008351207-000P 266429 CCUCUGCCGAUCCAUACUG CAGuAuGGAucGGcAGAGGUU 125 R-008351212-000N   5330 CAAGCCUCCAAGCUGUGCC B cAAGccuccAAGcuGuGccTT B 126 R-008351212-000N  53 30 CAAGCCUCCAAGCUGUGCC GGCAcAGcuuGGAGGcuuGUU 127 R-008351215-000P2674 31 UCCAUACUGCGGAACUCCU B UCCAUACUGCGGAACUCCUTT B 128R-008351215-000P 2674 31 UCCAUACUGCGGAACUCCU AGGAGUUCCGCAGUAUGGAUU 129R-008351218-000R 1651 32 CAGAGUCUAGACUCGUGGU B CAGAGUCUAGACUCGUGGUTT B130 R-008351218-000R 1651 32 CAGAGUCUAGACUCGUGGU ACCACGAGUCUAGACUCUGUU131 R-008351220-000N  565 33 AAGAAGAACUCCCUCGCCUB AAGAAGAAcucccucGccuTT B 132 R-008351220-000N  565 33AAGAAGAACUCCCUCGCCU AGGcGAGGGAGuucuucuuUU 133 R-008351223-000P  456 34GAGUGUGGAUUCGCACUCC B GAGUGUGGAUUCGCACUCCTT B 134 R-008351223-000P  45634 GAGUGUGGAUUCGCACUCC GGAGUGCGAAUCCACACUCUU 135 R-008351226-000R 165635 UCUAGACUCGUGGUGGACU B UCUAGACUCGUGGUGGACUTT B 136 R-008351226-000R1656 35 UCUAGACUCGUGGUGGACU AGUCCACCACGAGUCUAGAUU 137 R-008351232-000Y2663 10 UCCUCUGCCGAUCCAUACU B uccucuGccGAuccAuAcuTT B 138R-008351232-000Y 2663 10 UCCUCUGCCGAUCCAUACU AGUAuGGAucGGcAGAGGAUU 139R-008351235-000Z   53 30 CAAGCCUCCAAGCUGUGCC B CAAGCCUCCAAGCUGUGCCTT B140 R-008351235-000Z   53 30 CAAGCCUCCAAGCUGUGCC GGCACAGCUUGGAGGCUUGUU141 R-008351237-000S 1821 18 CCUGCUGCUAUGCCUCAUCB ccuGcuGcuAuGccucAucTT B 142 R-008351237-000S 1821 18CCUGCUGCUAUGCCUCAUC GAUGAGGcAuAGcAGcAGGUU 143 R-008351243-000Z 2665 23CUCUGCCGAUCCAUACUGC B CUCUGCCGAUCCAUACUGCTT B 144 R-008351243-000Z 266523 CUCUGCCGAUCCAUACUGC GCAGUAUGGAUCGGCAGAGUU 145 R-008351246-000A 165132 CAGAGUCUAGACUCGUGGU B cAGAGucuAGAcucGuGGuTT B 146 R-008351246-000A1651 32 CAGAGUCUAGACUCGUGGU ACCAcGAGucuAGAcucuGUU 147 R-008351248-000T1824 36 GCUGCUAUGCCUCAUCUUC B GcuGcuAuGccucAucuucTT B 148R-008351248-000T 1824 36 GCUGCUAUGCCUCAUCUUC GAAGAuGAGGcAuAGcAGcUU 149R-008351254-000A 2664 29 CCUCUGCCGAUCCAUACUG B CCUCUGCCGAUCCAUACUGTT B150 R-008351254-000A 2664 29 CCUCUGCCGAUCCAUACUG CAGUAUGGAUCGGCAGAGGUU151 R-008351257-000B 1656 35 UCUAGACUCGUGGUGGACUB ucuAGAcucGuGGuGGAcuTT B 152 R-008351257-000B 1656 35UCUAGACUCGUGGUGGACU AGUccAccAcGAGucuAGAUU 153 R-008351266-000K 1818  5CAUCCUGCUGCUAUGCCUC B cAuccuGcuGcuAuGccucTT B 154 R-008351266-000K 1818 5 CAUCCUGCUGCUAUGCCUC GAGGcAuAGcAGcAGGAuGUU 155 R-008351271-000J 165437 AGUCUAGACUCGUGGUGGA B AGucuAGAcucGuGGuGGATT B 156 R-008351271-000J1654 37 AGUCUAGACUCGUGGUGGA UCCAccAcGAGucuAGAcuUU 157 R-008351274-000K 455 28 GGAGUGUGGAUUCGCACUC B GGAGuGuGGAuucGcAcucTT B 158R-008351274-000K  455 28 GGAGUGUGGAUUCGCACUC GAGuGcGAAuccAcAcuccUU 159R-008351276-000C 1823  6 UGCUGCUAUGCCUCAUCUU B uGcuGcuAuGccucAucuuTT B160 R-008351276-000C 1823  6 UGCUGCUAUGCCUCAUCUU AAGAuGAGGcAuAGcAGcAUU161 R-008351281-000B 2674 31 UCCAUACUGCGGAACUCCUB uccAuAcuGcGGAAcuccuTT B 162 R-008351281-000B 2674 31UCCAUACUGCGGAACUCCU AGGAGuuccGcAGuAuGGAUU 163 R-008351284-000C 2662 38CUCCUCUGCCGAUCCAUAC B CUCCUCUGCCGAUCCAUACTT B 164 R-008351284-000C 266238 CUCCUCUGCCGAUCCAUAC GUAUGGAUCGGCAGAGGAGUU 165 R-008351287-000D 267224 GAUCCAUACUGCGGAACUC B GAuccAuAcuGcGGAAcucTT B 166 R-008351287-000D2672 24 GAUCCAUACUGCGGAACUC GAGuuccGcAGuAuGGAucUU 167 R-008351290-000K 567 25 GAAGAACUCCCUCGCCUCG B GAAGAAcucccucGccucGTT B 168R-008351290-000K  567 25 GAAGAACUCCCUCGCCUCG CGAGGcGAGGGAGuucuucUU 169R-008351292-000C 1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucATT B170 R-008351292-000C 1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUU171 R-008351294-000V 1820 20 UCCUGCUGCUAUGCCUCAUB uccuGcuGcuAuGccucAuTT B 172 R-008351294-000V 1820 20UCCUGCUGCUAUGCCUCAU AUGAGGcAuAGcAGcAGGAUU 173 R-008351309-000S 2673 22AUCCAUACUGCGGAACUCC B AUCCAUACUGCGGAACUCCTT B 174 R-008351309-000S 267322 AUCCAUACUGCGGAACUCC GGAGUUCCGCAGUAUGGAUUU 175 R-008351312-000Y 207739 UGGCUCAGUUUACUAGUGC B UGGCUCAGUUUACUAGUGCTT B 176 R-008351312-000Y2077 39 UGGCUCAGUUUACUAGUGC GCACUAGUAAACUGAGCCAUU 177 R-008351315-000Z  54 26 AAGCCUCCAAGCUGUGCCU B AAGCCUCCAAGCUGUGCCUTT B 178R-008351315-000Z   54 26 AAGCCUCCAAGCUGUGCCU AGGCACAGCUUGGAGGCUUUU 179R-008351320-000Y  566 27 AGAAGAACUCCCUCGCCUC B AGAAGAAcucccucGccucTT B180 R-008351320-000Y  566 27 AGAAGAACUCCCUCGCCUC GAGGcGAGGGAGuucuucuUU181 R-008351332-000H 1655 40 GUCUAGACUCGUGGUGGACB GucuAGAcucGuGGuGGAcTT B 182 R-008351332-000H 1655 40GUCUAGACUCGUGGUGGAC GUCcAccAcGAGucuAGAcUU 183 R-008351334-000A 1822 17CUGCUGCUAUGCCUCAUCU B cuGcuGcuAuGccucAucuTT B 184 R-008351334-000A 182217 CUGCUGCUAUGCCUCAUCU AGAuGAGGcAuAGcAGcAGUU 185 R-008351337-000B 266715 CUGCCGAUCCAUACUGCGG B cuGccGAuccAuAcuGcGGTT B 186 R-008351337-000B2667 15 CUGCCGAUCCAUACUGCGG CCGcAGuAuGGAucGGcAGUU 187 R-008351339-000U  51 41 UUCAAGCCUCCAAGCUGUG B uucAAGccuccAAGcuGuGTT B 188R-008351339-000U   51 41 UUCAAGCCUCCAAGCUGUG CACAGcuuGGAGGcuuGAAUU 189R-008351345-000B 1654 37 AGUCUAGACUCGUGGUGGA B AGUCUAGACUCGUGGUGGATT B190 R-008351345-000B 1654 37 AGUCUAGACUCGUGGUGGA UCCACCACGAGUCUAGACUUU191 R-008351348-000C 1655 40 GUCUAGACUCGUGGUGGACB GUCUAGACUCGUGGUGGACTT B 192 R-008351348-000C 1655 40GUCUAGACUCGUGGUGGAC GUCCACCACGAGUCUAGACUU 193 R-008351356-000C 2047 42CUAUGGGAGUGGGCCUCAG B CUAUGGGAGUGGGCCUCAGTT B 194 R-008351356-000C 204742 CUAUGGGAGUGGGCCUCAG CUGAGGCCCACUCCCAUAGUU 195 R-008351359-000D   5141 UUCAAGCCUCCAAGCUGUG B UUCAAGCCUCCAAGCUGUGTT B 196 R-008351359-000D  51 41 UUCAAGCCUCCAAGCUGUG CACAGCUUGGAGGCUUGAAUU 197 R-008351364-000C1660 21 GACUCGUGGUGGACUUCUC B GAcucGuGGuGGAcuucucTT B 198R-008351364-000C 1660 21 GACUCGUGGUGGACUUCUC GAGAAGuccAccAcGAGucUU 199R-008351370-000K 1824 36 GCUGCUAUGCCUCAUCUUC B GCUGCUAUGCCUCAUCUUCTT B200 R-008351370-000K 1824 36 GCUGCUAUGCCUCAUCUUC GAAGAUGAGGCAUAGCAGCUU201 R-008351374-000V 1664 16 CGUGGUGGACUUCUCUCAAB cGuGGuGGAcuucucucAATT B 202 R-008351374-000V 1664 16CGUGGUGGACUUCUCUCAA UUGAGAGAAGuccAccAcGUU 203 R-008351377-000W 2077 39UGGCUCAGUUUACUAGUGC B uGGcucAGuuuAcuAGuGcTT B 204 R-008351377-000W 207739 UGGCUCAGUUUACUAGUGC GCAcuAGuAAAcuGAGccAUU 205 R-008351380-000C 266238 CUCCUCUGCCGAUCCAUAC B cuccucuGccGAuccAuAcTT B 206 R-008351380-000C2662 38 CUCCUCUGCCGAUCCAUAC GUAuGGAucGGcAGAGGAGUU 207 R-008351383-000D 456 34 GAGUGUGGAUUCGCACUCC B GAGuGuGGAuucGcAcuccTT B 208R-008351383-000D  456 34 GAGUGUGGAUUCGCACUCC GGAGuGcGAAuccAcAcucUU 209R-008351392-000M 2934 14 CGCACCUCUCUUUACGCGG B cGcAccucucuuuAcGcGGTT B210 R-008351392-000M 2934 14 CGCACCUCUCUUUACGCGG CCGcGuAAAGAGAGGuGcGUU211 R-008351395-000N 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACTT B 212 R-008351395-000N 2670  4CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUU 213 R-008351398-000P 2047 42CUAUGGGAGUGGGCCUCAG B cuAuGGGAGuGGGccucAGTT B 214 R-008351398-000P 204742 CUAUGGGAGUGGGCCUCAG CUGAGGcccAcucccAuAGUU 215 R-008351401-000G  56533 AAGAAGAACUCCCUCGCCU B AAGAAGAACUCCCUCGCCUTT B 216 R-008351401-000G 565 33 AAGAAGAACUCCCUCGCCU AGGCGAGGGAGUUCUUCUUUU 217 R-008351404-000H2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAATT B 218R-008351404-000H 2669  3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUU 219R-008380305-000E 2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B220 R-008380305-000E 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU221 R-008380308-000F 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 222 R-008380308-000F 2670  4CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223 R-008380311-000M 2669  3GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224 R-008380311-000M 2669 3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 225 R-008380313-000E 2669 3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226 R-008380313-000E2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 225 R-008380315-000X2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224R-008380315-000X 2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 227R-008380316-000F 2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B226 R-008380316-000F 2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU227 R-008380318-000Y 2669  3 GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU228 R-008380318-000Y 2669  3 GCCGAUCCAUACUGCGGAAB GCCGAUCCAUACUGCGGAAUsU B 226 R-008380321-000E 2669  3GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAA UsU B 229 R-008380321-000E2669  3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU 230 R-008380324-000F2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 231R-008380324-000F 2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 232R-008380327-000G 1665  2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B233 R-008380327-000G 1665  2 GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU234 R-008380330-000N 1665  2 GUGGUGGACUUCUCUCAAUB GUGGUGGACUUCUCUCAAUUsU B 235 R-008380330-000N 1665  2GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 236 R-008380333-000P 1663  1UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237 R-008380333-000P 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 238 R-008380335-000G 1663 1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237 R-008380335-000G1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUsU 239 R-008380337-000Z1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237R-008380337-000Z 1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 240R-008380340-000F 1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B241 R-008380340-000F 1663  1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU242 R-008380343-000G 1663  1 UCGUGGUGGACUUCUCUCA uGAGAGAAGuccAccAcGAUsU243 R-008380343-000G 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 244 R-008380346-000H 2670  4CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 245 R-008380346-000H 2670 4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246 R-008380348-000A 2669 3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380348-000A2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 225 R-008380349-000J2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224R-008380349-000J 2669  3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU 230R-008380351-000G 2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B224 R-008380351-000G 2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU248 R-008380352-000R 2669  3 GCCGAUCCAUACUGCGGAAB GCCGAUCCAUACUGCGGAAUsU B 247 R-008380352-000R 2669  3GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 248 R-008380353-000Z 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380353-000Z 2669 3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 227 R-008380354-000H 2669 3 GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU 228 R-008380354-000H 2669 3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224 R-008380356-000A2669  3 GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU 228 R-008380356-000A2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 249R-008380359-000B 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B250 R-008380359-000B 1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU251 R-008380361-000Z 1665  2 GUGGUGGACUUCUCUCAAUB GuGGuGGAcuucucucAAuUsU B 252 R-008380361-000Z 1665  2GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU 234 R-008380362-000H 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380362-000H 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 238 R-008380363-000S 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380363-000S1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 240 R-008380365-000J1663  1 UCGUGGUGGACUUCUCUCA uGAGAGAAGuccAccAcGAUsU 243 R-008380365-000J1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 253R-008380367-000B 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B222 R-008380367-000B 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU254 R-008380369-000U 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 222 R-008380369-000U 2670  4CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 255 R-008380370-000H 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226 R-008380370-000H 2669 3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 248 R-008380372-000A 1665 2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380372-000A1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 256 R-008380374-000T1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257R-008380374-000T 1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 251R-008380377-000U 1665  2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B258 R-008380377-000U 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU259 R-008380378-000C 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 244 R-008380378-000C 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUsU 239 R-008380379-000L 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380379-000L 1663 1 UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUsU 239 R-008380381-000J 1663 1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237 R-008380381-000J1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 260 R-008380383-000B2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 261R-008380383-000B 2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 255R-008380384-000K 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B261 R-008380384-000K 2670  4 CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU223 R-008380386-000C 2670  4 CCGAUCCAUACUGCGGAACB ccGAuccAuAcuGcGGAAcUsU B 262 R-008380386-000C 2670  4CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223 R-008380387-000L 2669  3GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU 228 R-008380387-000L 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380389-000D 1665 2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 250 R-008380389-000D1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 263 R-008380390-000T1665  2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233R-008380390-000T 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU 259R-008380391-000B 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B250 R-008380391-000B 1665  2 GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU234 R-008380394-000C 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 264 R-008380394-000C 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 265 R-008380396-000V 2670  4CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 222 R-008380396-000V 2670 4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 266 R-008380397-000D 2670 4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 220 R-008380397-000D2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246 R-008380398-000M2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 261R-008380398-000M 2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246R-008380399-000W 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B222 R-008380399-000W 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU221 R-008380401-000E 2669  3 GCCGAUCCAUACUGCGGAAB GccGAuccAuAcuGcGGAAUsU B 224 R-008380401-000E 2669  3GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 267 R-008380402-000N 1665  2GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 250 R-008380402-000N 1665 2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU 259 R-008380404-000F 1665 2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380404-000F1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 268 R-008380405-000P1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257R-008380405-000P 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 268R-008380406-000Y 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B250 R-008380406-000Y 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU268 R-008380407-000G 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 241 R-008380407-000G 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 260 R-008380408-000R 1663  1UCGUGGUGGACUUCUCUCA uGAGAGAAGuccAccAcGAUsU 243 R-008380408-000R 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380409-000Z 2670 4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 220 R-008380409-000Z2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 266 R-008380410-000N2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 222R-008380410-000N 2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246R-008380411-000X 2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B220 R-008380411-000X 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU254 R-008380412-000F 2670  4 CCGAUCCAUACUGCGGAACB ccGAuccAuAcuGcGGAAcUsU B 220 R-008380412-000F 2670  4CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 255 R-008380413-000P 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380413-000P 2669 3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU 230 R-008380414-000Y 2669 3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226 R-008380414-000Y2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 267 R-008380415-000G1665  2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233R-008380415-000G 1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 263R-008380416-000R 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B257 R-008380416-000R 1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU263 R-008380417-000Z 1665  2 GUGGUGGACUUCUCUCAAUB GUGGUGGACUUCUCUCAAUUsU B 250 R-008380417-000Z 1665  2GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 256 R-008380418-000H 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380418-000H 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 242 R-008380420-000F 1663 1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucA UsU B 269 R-008380420-000F1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUsU 239 R-008380421-000P2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 261R-008380421-000P 2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 266R-008380422-000Y 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B261 R-008380422-000Y 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU254 R-008380423-000G 2670  4 CCGAUCCAUACUGCGGAACB ccGAuccAuAcuGcGGAAcUsU B 220 R-008380423-000G 2670  4CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223 R-008380426-000H 2670  4CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 270 R-008380426-000H 2670 4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU 271 R-008380427-000S 2669 3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380427-000S2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 267 R-008380428-000A1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257R-008380428-000A 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU 259R-008380429-000J 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B257 R-008380429-000J 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU256 R-008380430-000Y 1665  2 GUGGUGGACUUCUCUCAAUB GuGGuGGAcuucucucAAuUsU B 233 R-008380430-000Y 1665  2GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 251 R-008380431-000G 1665  2GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257 R-008380431-000G 1665 2 GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU 234 R-008380432-000R 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380432-000R1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 240 R-008380433-000Z1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237R-008380433-000Z 1663  1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 242R-008380434-000H 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B261 R-008380434-000H 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU221 R-008380435-000S 2669  3 GCCGAUCCAUACUGCGGAAB GCCGAUCCAUACUGCGGAAUsU B 226 R-008380435-000S 2669  3GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU 230 R-008380436-000A 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380436-000A 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 238 R-008380437-000J 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380437-000J1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 260 R-008380438-000T1663  1 UCGUGGUGGACUUCUCUCA uGAGAGAAGuccAccAcGAUsU 243 R-008380438-000T1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237R-008380441-000Z 2670  4 CCGAUCCAUACUGCGGAAC B CCGAU CCA UaCUgCggAACUsU B 272 R-008380441-000Z 2670  4 CCGAUCCAUACUGCGGAACGUUcCgcaGuAUggAuCgGUsU 273 R-008380444-000A 2670  4 CCGAUCCAUACUGCGGAACB CCgAUCCaUaCUgcGgAaC UsU B 274 R-008380444-000A 2670  4CCGAUCCAUACUGCGGAAC GuucCgCAgUAUGgAUCgGUsU 275 R-008380447-000B 2670  4CCGAUCCAUACUGCGGAAC gUuCcgCaGuauGgaUcGGUsU 276 R-008380447-000B 2670  4CCGAUCCAUACUGCGGAAC B cCGAUC CauA CUGCgGA ACUsU B 277 R-008380450-000H2669  3 GCCGAUCCAUACUGCGGAA uUccGCAGuaUgGaUCgGCUsU 278 R-008380450-000H2669  3 GCCGAUCCAUACUGCGGAA B GCCgaUC CaU ACUGCgGaA UsU B 279R-008380453-000J 1665  2 GUGGUGGACUUCUCUCAAU B GUGGU GgA C UUcU CUCAAUUsU B 280 R-008380453-000J 1665  2 GUGGUGGACUUCUCUCAAUAuugAgAgAAgUCcACCACUsU 281 R-008380456-000K 1665  2 GUGGUGGACUUCUCUCAAUaUUgAGAgAAgUCcACcaCUsU 282 R-008380456-000K 1665  2 GUGGUGGACUUCUCUCAAUB GUGgUgGaCUUCUCUCAA UUsU B 283 R-008380459-000L 1665  2GUGGUGGACUUCUCUCAAU B GUGgUGgaC U U CUCUCaaU UsU B 284 R-008380459-000L1665  2 GUGGUGGACUUCUCUCAAU AUugaGaGaAGuCcACCACUsU 285 R-008380462-000T1663  1 UCGUGGUGGACUUCUCUCA B UCG U GGUggaCuUCUCU CaUsU B 286R-008380462-000T 1663  1 UCGUGGUGGACUUCUCUCA UGaGAGAaGUcCacCaCgAUsU 287R-008380465-000U 2670  4 CCGAUCCAUACUGCGGAAC gUuccGCAgUaUgGAuCggUsU 288R-008380465-000U 2670  4 CCGAUCCAUACUGCGGAAC B C CGaU CCAUaCuGCGgAAcUsU B 289 R-008380468-000V 2669  3 GCCGAUCCAUACUGCGGAAuUCcGcAgUAUGgAUcggCUsU 290 R-008380468-000V 2669  3 GCCGAUCCAUACUGCGGAAB G CCGAuCCAUaCuGCgGaaUsU B 291 R-008380471-000B 2669  3GCCGAUCCAUACUGCGGAA uUCcgCaGuAUggAUcGGCUsU 292 R-008380471-000B 2669  3GCCGAUCCAUACUGCGGAA B gcCG AU C CAUA CUG CGGAaUsU B 293 R-008380474-000C2669  3 GCCGAUCCAUACUGCGGAA B GcCGA UCCAUAC U GCGGaA UsU B 294R-008380474-000C 2669  3 GCCGAUCCAUACUGCGGAA UUCcGCagUaUgGAuCGGcUsU 295R-008380477-000D 1665  2 GUGGUGGACUUCUCUCAAU B GUgGUG GaCUU CUCUcaaUUsU B 296 R-008380477-000D 1665  2 GUGGUGGACUUCUCUCAAUAuUGaGaGaAgUcCACcacUsU 297 R-008380480-000K 1665  2 GUGGUGGACUUCUCUCAAUaUuGAGAgaAgUccACCACUsU 298 R-008380480-000K 1665  2 GUGGUGGACUUCUCUCAAUB GUG GUggAC UuCU C UCA AU UsU B 299 R-008380483-000L 1665  2GUGGUGGACUUCUCUCAAU B gU G GUG GAC U U C UcUCaAU UsU B 300R-008380483-000L 1665  2 GUGGUGGACUUCUCUCAAU AUuGAGAGAAgUCCAcCaCUsU 301R-008380486-000M 1663  1 UCGUGGUGGACUUCUCUCA B U CgUGGUGGaCUU CuCU CAUsU B 302 R-008380486-000M 1663  1 UCGUGGUGGACUUCUCUCAUGaGagAAguccAcCACgaUsU 303 R-008380489-000N 1663  1 UCGUGGUGGACUUCUCUCAuGaGAGaAguCcacCACgAUsU 304 R-008380489-000N 1663  1 UCGUGGUGGACUUCUCUCAB uC GUggugGACUU CUCuCAUsU B 305 R-008380492-000V 1663  1UCGUGGUGGACUUCUCUCA ugAGaGAaGUCcaCcaCGAUsU 306 R-008380492-000V 1663  1UCGUGGUGGACUUCUCUCA B UCGuggUGgaCU UCucUCA UsU B 307 R-008380495-000W2670  4 CCGAUCCAUACUGCGGAAC B CcG AUCCAUACUgcGgaACUsU B 308R-008380495-000W 2670  4 CCGAUCCAUACUGCGGAAC GuuCcGCaGUAUGgauCGGUsU 309R-008380498-000X 2670  4 CCGAUCCAUACUGCGGAAC B C C G AUCCAUaCuGC GGAaCUsU B 310 R-008380498-000X 2670  4 CCGAUCCAUACUGCGGAACGUUCCGCAGUAUgGAUcgGUsU 311 R-008380501-000P 2670  4 CCGAUCCAUACUGCGGAACB CC G A UCCAUACUG CG GAaC UsU B 312 R-008380501-000P 2670  4CCGAUCCAUACUGCGGAAC GUUcCGCagUaugGAUcGgUsU 313 R-008380504-000R 2670  4CCGAUCCAUACUGCGGAAC gUUCcgCAGUaUGgAuCGGUsU 314 R-008380504-000R 2670  4CCGAUCCAUACUGCGGAAC B CCGaUCCAU ACUG CggaA CUsU B 315 R-008380507-000S2669  3 GCCGAUCCAUACUGCGGAA B GC CgaUCCAUACUgCG GaaUsU B 316R-008380507-000S 2669  3 GCCGAUCCAUACUGCGGAA UUCCgcAGUaUggauCGGcUsU 317R-008380510-000Y 1665  2 GUGGUGGACUUCUCUCAAU auUgAGAGAaGUccacCaCUsU 318R-008380510-000Y 1665  2 GUGGUGGACUUCUCUCAAU B GUggUggA CUUCuCuCaaUUsU B319 R-008380513-000Z 1665  2 GUGGUGGACUUCUCUCAAU B gUggUggaCUUCUCUCaaUUsU B 320 R-008380513-000Z 1665  2 GUGGUGGACUUCUCUCAAUAUUGAgAgaaGUccacCACUsU 321 R-008380516-000A 1665  2 GUGGUGGACUUCUCUCAAUB gU G G U G GaCUUC U C U C AaU UsU B 322 R-008380516-000A 1665  2GUGGUGGACUUCUCUCAAU AuuGAgaGAaGUCcACCaCUsU 323 R-008380519-000B 2670  4CCGAUCCAUACUGCGGAAC B C CgaUCCAuacUgcggaACUsU B 324 R-008380519-000B2670  4 CCGAUCCAUACUGCGGAAC GUuCCGcAGUaUGgauCggUsU 325 R-008380522-000H2669  3 GCCGAUCCAUACUGCGGAA B gCCgaUCCaUaCUgCggaaUsU B 326R-008380522-000H 2669  3 GCCGAUCCAUACUGCGGAA UUCcGCagUAuGGaUcGgCUsU 327R-008380525-000J 2669  3 GCCGAUCCAUACUGCGGAA uUcCgCAGUAuGGaUCGgCUsU 328R-008380525-000J 2669  3 GCCGAUCCAUACUGCGGAA B GccgaUCCaUAC UGCgGAAUsU B 329 R-008380528-000K 2669  3 GCCGAUCCAUACUGCGGAAuucCgCAGuAUgGaUCGGCUsU 330 R-008380528-000K 2669  3 GCCGAUCCAUACUGCGGAAB GCC GAuCCaUACUgCGGAAUsU B 331 R-008380531-000S 1665  2GUGGUGGACUUCUCUCAAU aUUgagAGAAguCcACCAcUsU 332 R-008380531-000S 1665  2GUGGUGGACUUCUCUCAAU B gUggUggAcuUC UCUCAaUUsU B 333 R-008380534-000T1663  1 UCGUGGUGGACUUCUCUCA uGAGAgAAgUCCacCACGAUsU 334 R-008380534-000T1663  1 UCGUGGUGGACUUCUCUCA B UCGUG GUggACuuCU C UC AUsU B 335R-008380537-000U 1663  1 UCGUGGUGGACUUCUCUCA B UCgUgGUgGAC UUC UCUCAUsU B 336 R-008380537-000U 1663  1 UCGUGGUGGACUUCUCUCAUGagAgAaGuCCAccaCGaUsU 337 R-008380540-000A 1663  1 UCGUGGUGGACUUCUCUCAB U CgugG U G G ACU UCUCUcA UsU B 338 R-008380540-000A 1663  1UCGUGGUGGACUUCUCUCA UgAGAgAagUCCAcCACGAUsU 339 R-008380543-000B 1663  1UCGUGGUGGACUUCUCUCA B UC GU GGUGG ACUUC U C UcaUsU B 340R-008380543-000B 1663  1 UCGUGGUGGACUUCUCUCA UgagagAAGucCACCAcgAUsU 341R-008380546-000C 2670  4 CCGAUCCAUACUGCGGAAC gUuCcGCAgUAUggAUcgGUsU 342R-008380546-000C 2670  4 CCGAUCCAUACUGCGGAAC B CC GaUC C A U ACUGCGgaA CUsU B 343 R-008380549-000D 2670  4 CCGAUCCAUACUGCGGAACguUCCGcAGuAUggaUCGgUsU 344 R-008380549-000D 2670  4 CCGAUCCAUACUGCGGAACB CCgA UCcaUaCugC GGA ACUsU B 345 R-008380552-000K 2669  3GCCGAUCCAUACUGCGGAA B GcCgA UCCAUaCUgcGgaA UsU B 346 R-008380552-000K2669  3 GCCGAUCCAUACUGCGGAA UuCCgCAGUAugGaUcGGCUsU 347 R-008380555-000L2669  3 GCCGAUCCAUACUGCGGAA B GCCGaU CC A U ACU GcGGAaUsU B 348R-008380555-000L 2669  3 GCCGAUCCAUACUGCGGAA UucCgCAGUAuGgaUCgGcUsU 349R-008380558-000M 1665  2 GUGGUGGACUUCUCUCAAU auuGAGaGAagUCCAcCACUsU 350R-008380558-000M 1665  2 GUGGUGGACUUCUCUCAAU B G UgGU G GAcUUC U CuC AaUUsU B 351 R-008380561-000U 1665  2 GUGGUGGACUUCUCUCAAU B gUGgUG GACUU CUC UCA AUUsU B 352 R-008380561-000U 1665  2 GUGGUGGACUUCUCUCAAUAUuGaGAGAaGUCcacCACUsU 353 R-008380564-000V 1663  1 UCGUGGUGGACUUCUCUCAugagaGAAGUCcACcACgaUsU 354 R-008380564-000V 1663  1 UCGUGGUGGACUUCUCUCAB UC GUG GUGgaCUU CUCU C A UsU B 355 R-008380567-000W 2669  3GCCGAUCCAUACUGCGGAA B GCcgaUCC A UaCUgC GGaA UsU B 356 R-008380567-000W2669  3 GCCGAUCCAUACUGCGGAA UucCGCaGUaUGGauCGGCUsU 357 R-008380570-000C1665  2 GUGGUGGACUUCUCUCAAU B GuGgUgGACUUcUCUcaA UUsU B 358R-008380570-000C 1665  2 GUGGUGGACUUCUCUCAAU AUUgagAgAAGUcCAcCAcUsU 359R-008380573-000D 1665  2 GUGGUGGACUUCUCUCAAU aUUGAgAgaaGuCCaCcAcUsU 360R-008380573-000D 1665  2 GUGGUGGACUUCUCUCAAU B gU GguGgaCUuCUCU CAauUsU B 361 R-008380576-000E 1665  2 GUGGUGGACUUCUCUCAAUauUGagaGaAGUCcaCcAcUsU 362 R-008380576-000E 1665  2 GUGGUGGACUUCUCUCAAUB gUgGuggACU UCUCUCaAU UsU B 363 R-008380579-000F 1663  1UCGUGGUGGACUUCUCUCA B UCGUGgUggA CU UCU CUCA UsU B 364 R-008380579-000F1663  1 UCGUGGUGGACUUCUCUCA UGAgAGaAguCCAcCACGaUsU 365 R-008380582-000M1663  1 UCGUGGUGGACUUCUCUCA B UCgUggUGgA C UUCU C UcaUsU B 366R-008380582-000M 1663  1 UCGUGGUGGACUUCUCUCA UGAgagaAGUcCAcCAcGAUsU 367R-008380585-000N 2670  4 CCGAUCCAUACUGCGGAAC B C C GAUCCaUaCUgCgGAaCUsU B 368 R-008380585-000N 2670  4 CCGAUCCAUACUGCGGAACGuuCCgCAGUAuggAUCGgUsU 369 R-008380588-000P 2670  4 CCGAUCCAUACUGCGGAACguUcCGCAguAuGgaUCGGUsU 370 R-008380588-000P 2670  4 CCGAUCCAUACUGCGGAACB C CgA UcCAuA CUGCgGaAC UsU B 371 R-008380591-000W 2670  4CCGAUCCAUACUGCGGAAC gUUCcgCAGUAuggaUCgGUsU 372 R-008380591-000W 2670  4CCGAUCCAUACUGCGGAAC B CcGaUC CA UaCU GCGG AaC UsU B 373 R-008380594-000X2669  3 GCCGAUCCAUACUGCGGAA B gC CgAUCC A UaCUgCgGA AUsU B 374R-008380594-000X 2669  3 GCCGAUCCAUACUGCGGAA UuCCGCAgUAUGGaUCGGcUsU 375R-008380597-000Y 2669  3 GCCGAUCCAUACUGCGGAA uuCCgcagUauGGaUCGGCUsU 376R-008380597-000Y 2669  3 GCCGAUCCAUACUGCGGAA B GCC GAUC C AU A CUgCGgaaUsU B 377 R-008380600-000R 2669  3 GCCGAUCCAUACUGCGGAA B GCCG AUCcauA C UgCGgaA UsU B 378 R-008380600-000R 2669  3 GCCGAUCCAUACUGCGGAAUUCCGcAGUaUGgaucGgCUsU 379 R-008380603-000S 1663  1 UCGUGGUGGACUUCUCUCAB UCgUggUggaCUUCUCUCaUsU B 380 R-008380603-000S 1663  1UCGUGGUGGACUUCUCUCA UgaGAGAagUCcacCACgAUsU 381 R-008380606-000T 1663  1UCGUGGUGGACUUCUCUCA uGAgAgAAguCCACCacGAUsU 382 R-008380606-000T 1663  1UCGUGGUGGACUUCUCUCA B ucgUgGUgGaCUuCU CUCaUsU B 383 R-008380609-000U2670  4 CCGAUCCAUACUGCGGAAC guUccGCaGUAUggAUCGgUsU 384 R-008380609-000U2670  4 CCGAUCCAUACUGCGGAAC B C CgAUcCaUACUgCGgAAC UsU B 385R-008380612-000A 2670  4 CCGAUCCAUACUGCGGAAC gUUCCGCagUAuGgaucGGUsU 386R-008380612-000A 2670  4 CCGAUCCAUACUGCGGAAC B CCgAUCCAUACUG CGgaaCUsU B 387 R-008380615-000B 2670  4 CCGAUCCAUACUGCGGAACgUUccGCAGUaUGgAUCGgUsU 388 R-008380615-000B 2670  4 CCGAUCCAUACUGCGGAACB CCgaUCCaUaCUgCggaaC UsU B 389 R-008380618-000C 2670  4CCGAUCCAUACUGCGGAAC B cCGaUCCaUA CU GCggA ACUsU B 390 R-008380618-000C2670  4 CCGAUCCAUACUGCGGAAC GuUCcgCAGuaugGaUCgGUsU 391 R-008380621-000J2669  3 GCCGAUCCAUACUGCGGAA B gCCGaUCCaUACUG CggAaUsU B 392R-008380621-000J 2669  3 GCCGAUCCAUACUGCGGAA UUcCgCAGUauGGAUcggCUsU 393R-008380624-000K 2669  3 GCCGAUCCAUACUGCGGAA B gCCGaUCCaUaC UgCGgAAUsU B 394 R-008380624-000K 2669  3 GCCGAUCCAUACUGCGGAAUUCCGCaGuAUGgaucgGCUsU 395 R-008380627-000L 2669  3 GCCGAUCCAUACUGCGGAAuUCCGCAGUAUGGaUcggcUsU 396 R-008380627-000L 2669  3 GCCGAUCCAUACUGCGGAAB G C C GAU C C AUACUGCgGAA UsU B 397 R-008380630-000T 2669  3GCCGAUCCAUACUGCGGAA B GCCgAUC CAU ACUGCG GAAUsU B 398 R-008380630-000T2669  3 GCCGAUCCAUACUGCGGAA UUcCgCAGuAuggauCGGCUsU 399 R-008380633-000U1665  2 GUGGUGGACUUCUCUCAAU B GugGUGgaC UU CU C UcAAUUsU B 400R-008380633-000U 1665  2 GUGGUGGACUUCUCUCAAU AuUGAGaGaaGUCCAccaCUsU 401R-008380636-000V 1665  2 GUGGUGGACUUCUCUCAAU B G UGGUGG ACU UcUcUCaaUUsU B 402 R-008380636-000V 1665  2 GUGGUGGACUUCUCUCAAUAuUGAgAGAAgucCaCCACUsU 403 R-008380639-000W 1665  2 GUGGUGGACUUCUCUCAAUaUUGAGAgaAguccACCaCUsU 404 R-008380639-000W 1665  2 GUGGUGGACUUCUCUCAAUB GU GgUgGaCU U CUCUCAA U UsU B 405 R-008380642-000C 1663  1UCGUGGUGGACUUCUCUCA B UCgUGgUgGACUUcUcUCaUsU B 406 R-008380642-000C 1663 1 UCGUGGUGGACUUCUCUCA UgAGAgAAGuCCAcCaCGaUsU 407 R-008380645-000D 1663 1 UCGUGGUGGACUUCUCUCA B UCgUggUgGAcUU C UCUCA UsU B 408R-008380645-000D 1663  1 UCGUGGUGGACUUCUCUCA UgaGAGaAGUCCAcCacGaUsU 409R-008380648-000E 1663  1 UCGUGGUGGACUUCUCUCA ugAGaGaaGuCCAcCaCGAUsU 410R-008380648-000E 1663  1 UCGUGGUGGACUUCUCUCA B UCG UgguGGaC UUcUCUCaUsU B 411 R-008380651-000L 1663  1 UCGUGGUGGACUUCUCUCA B uCGuGGUgGaCUU CUCUcA UsU B 412 R-008380651-000L 1663  1 UCGUGGUGGACUUCUCUCAUgaGagaAGUcCacCAcGaUsU 413 R-008380654-000M 1663  1 UCGUGGUGGACUUCUCUCAuGagAGAAgucCACCACGaUsU 414 R-008380654-000M 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGgUGgACUuCuC UCAUsU B 415 R-008380655-000W 1665  2GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 250 R-008380655-000W 1665 2 GUGGUGGACUUCUCUCAAU AUuGAGAGAAgUCCAcCaCUsU 301 R-008380656-000E 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380656-000E1663  1 UCGUGGUGGACUUCUCUCA UgAGAgAagUCCAcCACGAUsU 339 R-008380657-000N2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 222R-008380657-000N 2670  4 CCGAUCCAUACUGCGGAAC GUUcCgcaGuAUggAuCgGUsU 273R-008380658-000X 2670  4 CCGAUCCAUACUGCGGAAC gUuccGCAgUaUgGAuCggUsU 288R-008380658-000X 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B261 R-008380659-000F 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 261 R-008380659-000F 2670  4CCGAUCCAUACUGCGGAAC GUuCCGcAGUaUGgauCggUsU 325 R-008380660-000V 2669  3GCCGAUCCAUACUGCGGAA uUccGCAGuaUgGaUCgGCUsU 278 R-008380660-000V 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226 R-008380661-000D 1665 2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380661-000D1665  2 GUGGUGGACUUCUCUCAAU AuUGaGaGaAgUcCACcacUsU 297 R-008380662-000M1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237R-008380662-000M 1663  1 UCGUGGUGGACUUCUCUCA UgaGAGAagUCcacCACgAUsU 381R-008380663-000W 1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B237 R-008380663-000W 1663  1 UCGUGGUGGACUUCUCUCA UGAgAGaAguCCAcCACGaUsU365 R-008380664-000E 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 244 R-008380664-000E 1663  1UCGUGGUGGACUUCUCUCA UGaGagAAguccAcCACgaUsU 303 R-008380665-000N 1663  1UCGUGGUGGACUUCUCUCA ugAGaGaaGuCCAcCaCGAUsU 410 R-008380665-000N 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380666-000X 1663 1 UCGUGGUGGACUUCUCUCA uGaGAGaAguCcacCACgAUsU 304 R-008380666-000X 1663 1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237 R-008380667-000F1663  1 UCGUGGUGGACUUCUCUCA uGagAGAAgucCACCACGaUsU 414 R-008380667-000F1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241R-008380668-000P 2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B224 R-008380668-000P 2669  3 GCCGAUCCAUACUGCGGAA UUCCGCaGuAUGgaucgGCUsU395 R-008380669-000Y 2669  3 GCCGAUCCAUACUGCGGAAB GCCGAUCCAUACUGCGGAAUsU B 226 R-008380669-000Y 2669  3GCCGAUCCAUACUGCGGAA UUCCgcAGUaUggauCGGcUsU 317 R-008380670-000M 1665  2GUGGUGGACUUCUCUCAAU aUUGAGAgaAguccACCaCUsU 404 R-008380670-000M 1665  2GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257 R-008380671-000W 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380671-000W1663  1 UCGUGGUGGACUUCUCUCA UgagagAAGucCACCAcgAUsU 341 R-008380672-000E1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237R-008380672-000E 1663  1 UCGUGGUGGACUUCUCUCA UgaGAGaAGUCCAcCacGaUsU 409R-008380673-000N 1663  1 UCGUGGUGGACUUCUCUCA uGAgAgAAguCCACCacGAUsU 382R-008380673-000N 1663  1 UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B237 R-008380674-000X 2670  4 CCGAUCCAUACUGCGGAAC guUCCGcAGuAUggaUCGgUsU344 R-008380674-000X 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 222 R-008380675-000F 2669  3GCCGAUCCAUACUGCGGAA uUcCgCAGUAuGGaUCGgCUsU 328 R-008380675-000F 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380676-000P 2669 3 GCCGAUCCAUACUGCGGAA uUCcgCaGuAUggAUcGGCUsU 292 R-008380676-000P 2669 3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224 R-008380677-000Y2669  3 GCCGAUCCAUACUGCGGAA uucCgCAGuAUgGaUCGGCUsU 330 R-008380677-000Y2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226R-008380678-000G 1665  2 GUGGUGGACUUCUCUCAAU auUgAGAGAaGUccacCaCUsU 318R-008380678-000G 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B257 R-008380679-000R 1665  2 GUGGUGGACUUCUCUCAAU aUUGAgAgaaGuCCaCcAcUsU360 R-008380679-000R 1665  2 GUGGUGGACUUCUCUCAAUB GuGGuGGAcuucucucAAuUsU B 233 R-008380680-000E 1665  2GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 250 R-008380680-000E 1665 2 GUGGUGGACUUCUCUCAAU AuugAgAgAAgUCcACCACUsU 281 R-008380681-000N 2670 4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 222 R-008380681-000N2670  4 CCGAUCCAUACUGCGGAAC GuuCcGCaGUAUGgauCGGUsU 309 R-008380682-000X1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244R-008380682-000X 1663  1 UCGUGGUGGACUUCUCUCA UGaGAGAaGUcCacCaCgAUsU 287R-008380683-000F 1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B241 R-008380683-000F 1663  1 UCGUGGUGGACUUCUCUCA UgAGAgAAGuCCAcCaCGaUsU407 R-008380684-000P 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 222 R-008380684-000P 2670  4CCGAUCCAUACUGCGGAAC GuucCgCAgUAUGgAUCgGUsU 275 R-008380685-000Y 2670  4CCGAUCCAUACUGCGGAAC guUccGCaGUAUggAUCGgUsU 384 R-008380685-000Y 2670  4CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 261 R-008380686-000G 2669 3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224 R-008380686-000G2669  3 GCCGAUCCAUACUGCGGAA UucCGCaGUaUGGauCGGCUsU 357 R-008380687-000R2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247R-008380687-000R 2669  3 GCCGAUCCAUACUGCGGAA UuCCgCAGUAugGaUcGGCUsU 347R-008380688-000Z 2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B247 R-008380688-000Z 2669  3 GCCGAUCCAUACUGCGGAA UuCCGCAgUAUGGaUCGGcUsU375 R-008380689-000H 2669  3 GCCGAUCCAUACUGCGGAA uuCCgcagUauGGaUCGGCUsU376 R-008380689-000H 2669  3 GCCGAUCCAUACUGCGGAAB GccGAuccAuAcuGcGGAAUsU B 224 R-008380690-000X 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380690-000X 2669 3 GCCGAUCCAUACUGCGGAA UUCCGcAGUaUGgaucGgCUsU 379 R-008380691-000F 2670 4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 220 R-008380691-000F2670  4 CCGAUCCAUACUGCGGAAC GuuCCgCAGUAuggAUCGgUsU 369 R-008380692-000P1665  2 GUGGUGGACUUCUCUCAAU aUUgagAGAAguCcACCAcUsU 332 R-008380692-000P1665  2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233R-008380693-000Y 2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B220 R-008380693-000Y 2670  4 CCGAUCCAUACUGCGGAAC GUUcCGCagUaugGAUcGgUsU313 R-008380694-000G 2670  4 CCGAUCCAUACUGCGGAAC guUcCGCAguAuGgaUCGGUsU370 R-008380694-000G 2670  4 CCGAUCCAUACUGCGGAACB ccGAuccAuAcuGcGGAAcUsU B 220 R-008380695-000R 2670  4CCGAUCCAUACUGCGGAAC gUuCcgCaGuauGgaUcGGUsU 276 R-008380695-000R 2670  4CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 220 R-008380696-000Z 2669 3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B 224 R-008380696-000Z2669  3 GCCGAUCCAUACUGCGGAA UUCcGCagUAuGGaUcGgCUsU 327 R-008380697-000H2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 226R-008380697-000H 2669  3 GCCGAUCCAUACUGCGGAA UUcCgCAGUauGGAUcggCUsU 393R-008380698-000S 2669  3 GCCGAUCCAUACUGCGGAA B GccGAuccAuAcuGcGGAAUsU B224 R-008380698-000S 2669  3 GCCGAUCCAUACUGCGGAA UucCgCAGUAuGgaUCgGcUsU349 R-008380699-000A 1665  2 GUGGUGGACUUCUCUCAAUB GUGGUGGACUUCUCUCAAUUsU B 250 R-008380699-000A 1665  2GUGGUGGACUUCUCUCAAU AUUgagAgAAGUcCAcCAcUsU 359 R-008380700-000A 1665  2GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380700-000A 1665 2 GUGGUGGACUUCUCUCAAU AUUGAgAgaaGUccacCACUsU 321 R-008380701-000J 1665 2 GUGGUGGACUUCUCUCAAU aUUgAGAgAAgUCcACcaCUsU 282 R-008380701-000J 1665 2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257 R-008380702-000T1663  1 UCGUGGUGGACUUCUCUCA uGAGAgAAgUCCacCACGAUsU 334 R-008380702-000T1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244R-008380703-000B 1663  1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B241 R-008380703-000B 1663  1 UCGUGGUGGACUUCUCUCA UGagAgAaGuCCAccaCGaUsU337 R-008380704-000K 1663  1 UCGUGGUGGACUUCUCUCAB UCGUGGUGGACUUCUCUCAUsU B 244 R-008380704-000K 1663  1UCGUGGUGGACUUCUCUCA UgaGagaAGUcCacCAcGaUsU 413 R-008380705-000U 1663  1UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 241 R-008380705-000U 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaAGUcCAcCAcGAUsU 367 R-008380706-000C 1663 1 UCGUGGUGGACUUCUCUCA ugAGaGAaGUCcaCcaCGAUsU 306 R-008380706-000C 1663 1 UCGUGGUGGACUUCUCUCA B UCGUGGUGGACUUCUCUCAUsU B 244 R-008380707-000L2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 261R-008380707-000L 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUgGAUcgGUsU 311R-008380708-000V 2670  4 CCGAUCCAUACUGCGGAAC gUuCcGCAgUAUggAUcgGUsU 342R-008380708-000V 2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B220 R-008380709-000D 2670  4 CCGAUCCAUACUGCGGAAC gUUccGCAGUaUGgAUCGgUsU388 R-008380709-000D 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 222 R-008380710-000T 2669  3GCCGAUCCAUACUGCGGAA uUCcGcAgUAUGgAUcggCUsU 290 R-008380710-000T 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380711-000B 1665 2 GUGGUGGACUUCUCUCAAU auUGagaGaAGUCcaCcAcUsU 362 R-008380711-000B 1665 2 GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380712-000K1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257R-008380712-000K 1665  2 GUGGUGGACUUCUCUCAAU AUugaGaGaAGuCcACCACUsU 285R-008380713-000U 2670  4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B261 R-008380713-000U 2670  4 CCGAUCCAUACUGCGGAAC GuUCcgCAGuaugGaUCgGUsU391 R-008380714-000C 2670  4 CCGAUCCAUACUGCGGAAC gUUCcgCAGUAuggaUCgGUsU372 R-008380714-000C 2670  4 CCGAUCCAUACUGCGGAACB CCGAUCCAUACUGCGGAACUsU B 261 R-008380715-000L 2669  3GCCGAUCCAUACUGCGGAA uUCCGCAGUAUGGaUcggcUsU 396 R-008380715-000L 2669  3GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B 247 R-008380716-000V 1665 2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 250 R-008380716-000V1665  2 GUGGUGGACUUCUCUCAAU AuUGAgAGAAgucCaCCACUsU 403 R-008380717-000D1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257R-008380717-000D 1665  2 GUGGUGGACUUCUCUCAAU AUuGaGAGAaGUCcacCACUsU 353R-008380718-000M 1665  2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B250 R-008380718-000M 1665  2 GUGGUGGACUUCUCUCAAU AuuGAgaGAaGUCcACCaCUsU323 R-008380719-000W 1665  2 GUGGUGGACUUCUCUCAAU aUuGAGAgaAgUccACCACUsU298 R-008380719-000W 1665  2 GUGGUGGACUUCUCUCAAUB GUGGUGGACUUCUCUCAAUUsU B 250 R-008380720-000K 1663  1UCGUGGUGGACUUCUCUCA ugagaGAAGUCcACcACgaUsU 354 R-008380720-000K 1663  1UCGUGGUGGACUUCUCUCA B ucGuGGuGGAcuucucucAUsU B 237 R-008380721-000U 2670 4 CCGAUCCAUACUGCGGAAC gUUCcgCAGUaUGgAuCGGUsU 314 R-008380721-000U 2670 4 CCGAUCCAUACUGCGGAAC B CCGAUCCAUACUGCGGAACUsU B 222 R-008380722-000C2670  4 CCGAUCCAUACUGCGGAAC gUUCCGCagUAuGgaucGGUsU 386 R-008380722-000C2670  4 CCGAUCCAUACUGCGGAAC B ccGAuccAuAcuGcGGAAcUsU B 220R-008380723-000L 2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCCAUACUGCGGAAUsU B226 R-008380723-000L 2669  3 GCCGAUCCAUACUGCGGAA UUcCgCAGuAuggauCGGCUsU399 R-008380724-000V 2669  3 GCCGAUCCAUACUGCGGAAB GCCGAUCCAUACUGCGGAAUsU B 226 R-008380724-000V 2669  3GCCGAUCCAUACUGCGGAA UUCcGCagUaUgGAuCGGcUsU 295 R-008380725-000D 1665  2GUGGUGGACUUCUCUCAAU B GuGGuGGAcuucucucAAuUsU B 233 R-008380725-000D 1665 2 GUGGUGGACUUCUCUCAAU AuUGAGaGaaGUCCAccaCUsU 401 R-008380726-000M 1665 2 GUGGUGGACUUCUCUCAAU auuGAGaGAagUCCAcCACUsU 350 R-008380726-000M 1665 2 GUGGUGGACUUCUCUCAAU B GUGGUGGACUUCUCUCAAUUsU B 257 R-008380727-000W2669  3 GCCGAUCCAUACUGCGGAA B gC CgAUCC A UaCUgCgGA AUsU B 374R-008380727-000W 2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 227R-008380728-000E 2669  3 GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU 228R-008380728-000E 2669  3 GCCGAUCCAUACUGCGGAA B GCCGAUCcauA C UgCGgaAUsU B 378 R-008380729-000N 1665  2 GUGGUGGACUUCUCUCAAU B GUgGU G GAcUUCU CuC AaU UsU B 351 R-008380729-000N 1665  2 GUGGUGGACUUCUCUCAAUAUUGAGAGAAGuccAccAcUsU 259 R-008380730-000C 1665  2 GUGGUGGACUUCUCUCAAUB gU GguGgaCUuCUCU C AauUsU B 361 R-008380730-000C 1665  2GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 256 R-008380731-000L 1665  2GUGGUGGACUUCUCUCAAU B gU G GUG GAC U U C UcUCaAU UsU B 300R-008380731-000L 1665  2 GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU 234R-008380732-000V 1663  1 UCGUGGUGGACUUCUCUCA B UCGUG GUggACuuCU C UCAUsU B 335 R-008380732-000V 1663  1 UCGUGGUGGACUUCUCUCAUGAGAGAAGuccAccAcGAUsU 239 R-008380733-000D 1663  1 UCGUGGUGGACUUCUCUCAuGAGAGAAGuccAccAcGAUsU 243 R-008380733-000D 1663  1 UCGUGGUGGACUUCUCUCAB uC GUggugGACUU CUCuCAUsU B 305 R-008380734-000M 2670  4CCGAUCCAUACUGCGGAAC B C CGaU C CAUaCuGCGgAAcUsU B 289 R-008380734-000M2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246 R-008380735-000W2670  4 CCGAUCCAUACUGCGGAAC B cCGAUC CauA CUGCgGA ACUsU B 277R-008380735-000W 2670  4 CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223R-008380736-000E 2669  3 GCCGAUCCAUACUGCGGAA B gCCgaUCCaUaCUgCggaaUsU B326 R-008380736-000E 2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU225 R-008380737-000N 2669  3 GCCGAUCCAUACUGCGGAA B gCCGaUCCaUaC UgCGgAAUsU B 394 R-008380737-000N 2669  3 GCCGAUCCAUACUGCGGAAUUCCGCAGUAUGGAUCGGCUsU 267 R-008380738-000X 2669  3 GCCGAUCCAUACUGCGGAAuuccGcAGuAuGGAucGGcUsU 228 R-008380738-000X 2669  3 GCCGAUCCAUACUGCGGAAB GCC G AuCCaUACUgCGGAAUsU B 331 R-008380739-000F 1665  2GUGGUGGACUUCUCUCAAU B G UGGUGG ACU UcUcU CaaUUsU B 402 R-008380739-000F1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 263 R-008380740-000V1665  2 GUGGUGGACUUCUCUCAAU B GUGGU GgA C UUcU CU CAAUUsU B 280R-008380740-000V 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 256R-008380741-000D 1663  1 UCGUGGUGGACUUCUCUCA B UCgUggUgGAcUU CUCUCAUsU B 408 R-008380741-000D 1663  1 UCGUGGUGGACUUCUCUCAUGAGAGAAGUCCACCACGAUsU 260 R-008380742-000M 1663  1 UCGUGGUGGACUUCUCUCAB ucgUgGUgGaCUuCUCUCaUsU B 383 R-008380742-000M 1663  1UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 242 R-008380743-000W 2670  4CCGAUCCAUACUGCGGAAC B CC GaUC C A U ACUGCGgaA C UsU B 343R-008380743-000W 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU 254R-008380744-000E 2670  4 CCGAUCCAUACUGCGGAAC B CCgAUCCaUaCUgcGgAaCUsU B274 R-008380744-000E 2670  4 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU254 R-008380745-000N 2670  4 CCGAUCCAUACUGCGGAAC B CCgaUCCAuacUgcggaACUsU B 324 R-008380745-000N 2670  4 CCGAUCCAUACUGCGGAACGUUCCGCAGUAUGGAUCGGUsU 254 R-008380746-000X 2670  4 CCGAUCCAUACUGCGGAACB C CgAUcCaUACUgCGgAAC UsU B 385 R-008380746-000X 2670  4CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGGUsU 221 R-008380747-000F 2670  4CCGAUCCAUACUGCGGAAC B CcGaUC CA UaCU GCGGAaC UsU B 373 R-008380747-000F2670  4 CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223 R-008380748-000P1665  2 GUGGUGGACUUCUCUCAAU B GuGgUgGACUUcUCUcaA UUsU B 358R-008380748-000P 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU 259R-008380749-000Y 1665  2 GUGGUGGACUUCUCUCAAU B gUggUggaCUUCUCUCaaU UsU B320 R-008380749-000Y 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU268 R-008380750-000M 1665  2 GUGGUGGACUUCUCUCAAU B GUG GUggAC UuCU C UCAAU UsU B 299 R-008380750-000M 1665  2 GUGGUGGACUUCUCUCAAUAUUgagagaagUCCaCCaCUsU 251 R-008380751-000W 1663  1 UCGUGGUGGACUUCUCUCAB UCgUGgUgGACUUcUcUCaUsU B 406 R-008380751-000W 1663  1UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 238 R-008380752-000E 2670  4CCGAUCCAUACUGCGGAAC B CcG AUCCAUACUgcGgaACUsU B 308 R-008380752-000E2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 266 R-008380753-000N2670  4 CCGAUCCAUACUGCGGAAC B CC G A UCCAUACUG CG GAaC UsU B 312R-008380753-000N 2670  4 CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246R-008380754-000X 2670  4 CCGAUCCAUACUGCGGAAC B C CgA UcCAuA CUGCgGaACUsU B 371 R-008380754-000X 2670  4 CCGAUCCAUACUGCGGAACGUUCCGCAGUAUGGAUCGGUsU 221 R-008380755-000F 2669  3 GCCGAUCCAUACUGCGGAAB G CCGAuCCAUaCuGCgGaaUsU B 291 R-008380755-000F 2669  3GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 225 R-008380756-000P 2669  3GCCGAUCCAUACUGCGGAA B GCCgaUC CaU ACUGCgGaA UsU B 279 R-008380756-000P2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 225 R-008380757-000Y2669  3 GCCGAUCCAUACUGCGGAA B GCCgAUC CAU ACUGCG G AA UsU B 398R-008380757-000Y 2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 248R-008380758-000G 2669  3 GCCGAUCCAUACUGCGGAA uuccGcAGuAuGGAucGGcUsU 228R-008380758-000G 2669  3 GCCGAUCCAUACUGCGGAA B GCC GAUC C AU A CUgCGgaaUsU B 377 R-008380759-000R 1665  2 GUGGUGGACUUCUCUCAAUB GUGgUGgaC U UCUCUCaaU UsU B 284 R-008380759-000R 1665  2GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU 234 R-008380760-000E 1663  1UCGUGGUGGACUUCUCUCA B UC GU GGUGG ACUUC U C UcaUsU B 340R-008380760-000E 1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 240R-008380761-000N 2670  4 CCGAUCCAUACUGCGGAAC B CCgaUCCaUaCUgCggaaC UsU B389 R-008380761-000N 2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU255 R-008380762-000X 2669  3 GCCGAUCCAUACUGCGGAA B GCcgaUCC A UaCUgCGGaA UsU B 356 R-008380762-000X 2669  3 GCCGAUCCAUACUGCGGAAUUCcGcAGuAuGGAucGGcUsU 230 R-008380763-000F 1665  2 GUGGUGGACUUCUCUCAAUB gUGgUG GACUU C UC UCA AUUsU B 352 R-008380763-000F 1665  2GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 256 R-008380764-000P 1665  2GUGGUGGACUUCUCUCAAU B gUggUggAcuUC UCUCAaUUsU B 333 R-008380764-000P1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 251 R-008380765-000Y1665  2 GUGGUGGACUUCUCUCAAU B G U GgUgGaCU U CUCUCAA U UsU B 405R-008380765-000Y 1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 251R-008380766-000G 1663  1 UCGUGGUGGACUUCUCUCA B UCGUGgUggA CU UCU CUCAUsU B 364 R-008380766-000G 1663  1 UCGUGGUGGACUUCUCUCAUGAGAGAAGuccAccAcGAUsU 239 R-008380767-000R 1663  1 UCGUGGUGGACUUCUCUCAuGAGAGAAGuccAccAcGAUsU 243 R-008380767-000R 1663  1 UCGUGGUGGACUUCUCUCAB UCGuggUGgaCU UCucUCA UsU B 307 R-008380768-000Z 2670  4CCGAUCCAUACUGCGGAAC B C C GAUC CaUaCUgCgGAaCUsU B 368 R-008380768-000Z2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 266 R-008380769-000H2669  3 GCCGAUCCAUACUGCGGAA B GCCGaU CCAU ACU GcGGAaUsU B 348R-008380769-000H 2669  3 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 248R-008380770-000X 2669  3 GCCGAUCCAUACUGCGGAA B GccgaUCCaUAC U GCgGAAUsU B 329 R-008380770-000X 2669  3 GCCGAUCCAUACUGCGGAAUUCCGCAGUAUGGAUCGGCUsU 248 R-008380771-000F 1665  2 GUGGUGGACUUCUCUCAAUB gU G G U G GaCUUC U C U C AaU UsU B 322 R-008380771-000F 1665  2GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 268 R-008380772-000P 1663  1UCGUGGUGGACUUCUCUCA B UCgUggUggaCUUCUCUCaUsU B 380 R-008380772-000P 1663 1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 238 R-008380773-000Y 1663 1 UCGUGGUGGACUUCUCUCA B UCgUgGUgGAC UUC UCUCA UsU B 336R-008380773-000Y 1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGuccAccAcGAUsU 239R-008380774-000G 1663  1 UCGUGGUGGACUUCUCUCA B UC GUG GUGgaCUU CUCU C AUsU B 355 R-008380774-000G 1663  1 UCGUGGUGGACUUCUCUCAUGAGAGAAGUCCACCACGAUsU 240 R-008380775-000R 1663  1 UCGUGGUGGACUUCUCUCAB UCG UgguGGaC UUcUC UCaUsU B 411 R-008380775-000R 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 260 R-008380776-000Z 1663  1UCGUGGUGGACUUCUCUCA B UCgUggUGgA C UUCU C UcaUsU B 366 R-008380776-000Z1663  1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 242 R-008380777-000H1663  1 UCGUGGUGGACUUCUCUCA uGAGAGAAGuccAccAcGAUsU 243 R-008380777-000H1663  1 UCGUGGUGGACUUCUCUCA B UCGUGgUGgACUuCuC UCAUsU B 415R-008380778-000S 2670  4 CCGAUCCAUACUGCGGAAC B CCGaUCCAU ACUG CggaACUsU B 315 R-008380778-000S 2670  4 CCGAUCCAUACUGCGGAACGUUCCGCAGUAUGGAUCGGUsU 221 R-008380779-000A 2670  4 CCGAUCCAUACUGCGGAACB cCGaUCCaUA CU GCggA ACUsU B 390 R-008380779-000A 2670  4CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 255 R-008380780-000P 2669  3GCCGAUCCAUACUGCGGAA B GcCgA UCCAUaCUgcGgaA UsU B 346 R-008380780-000P2669  3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU 230 R-008380781-000Y2669  3 GCCGAUCCAUACUGCGGAA B gcCG AU C C A UA CUG CGGAaUsU B 293R-008380781-000Y 2669  3 GCCGAUCCAUACUGCGGAA UUCCgCagUaUggaUCggCUsU 227R-008380782-000G 2669  3 GCCGAUCCAUACUGCGGAA B GcCGA UCCAUAC U GCGGaAUsU B 294 R-008380782-000G 2669  3 GCCGAUCCAUACUGCGGAAUUCCgCagUaUggaUCggCUsU 227 R-008380783-000R 1665  2 GUGGUGGACUUCUCUCAAUB GugGUGgaC UU CU C UcAAUUsU B 400 R-008380783-000R 1665  2GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 263 R-008380784-000Z 1665  2GUGGUGGACUUCUCUCAAU B GUggUggA CUUCuCuCaaUUsU B 319 R-008380784-000Z1665  2 GUGGUGGACUUCUCUCAAU AUUgagagaagUCCaCCaCUsU 263 R-008380785-000H1665  2 GUGGUGGACUUCUCUCAAU B GUGgUgGaCUUCUCUCAA UUsU B 283R-008380785-000H 1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCACUsU 268R-008380786-000S 2670  4 CCGAUCCAUACUGCGGAAC B C C G AUCCAUaCuGC GGAaCUsU B 310 R-008380786-000S 2670  4 CCGAUCCAUACUGCGGAACGUUCCgCagUaUggaUCggUsU 266 R-008380787-000A 2670  4 CCGAUCCAUACUGCGGAACB CCGAU CCA UaCUgCggAAC UsU B 272 R-008380787-000A 2670  4CCGAUCCAUACUGCGGAAC GUUccGcAGuAuGGAucGGUsU 246 R-008380788-000J 2670  4CCGAUCCAUACUGCGGAAC B CCgAUCCAUACUG C GgaaCUsU B 387 R-008380788-000J2670  4 CCGAUCCAUACUGCGGAAC GUUCCgCagUaUggaUCggUsU 255 R-008380789-000T2670  4 CCGAUCCAUACUGCGGAAC B CCgA UCcaUaCugC GGA ACUsU B 345R-008380789-000T 2670  4 CCGAUCCAUACUGCGGAAC GuuccGcAGuAuGGAucGGUsU 223R-008380790-000G 2669  3 GCCGAUCCAUACUGCGGAA B gCCGaUCCaUACUG CggAaUsU B392 R-008380790-000G 2669  3 GCCGAUCCAUACUGCGGAA UUCcGcAGuAuGGAucGGcUsU230 R-008380791-000R 2669  3 GCCGAUCCAUACUGCGGAA B G C C GAU C CAUACUGCgGAA UsU B 397 R-008380791-000R 2669  3 GCCGAUCCAUACUGCGGAAUUCCGCAGUAUGGAUCGGCUsU 267 R-008380792-000Z 2669  3 GCCGAUCCAUACUGCGGAAB GC CgaUCCAUACUgCG GaaUsU B 316 R-008380792-000Z 2669  3GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGCUsU 267 R-008380793-000H 1665  2GUGGUGGACUUCUCUCAAU B GUgGUG GaCUU CU CUcaaUUsU B 296 R-008380793-000H1665  2 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGuccAccAcUsU 259 R-008380794-000S1665  2 GUGGUGGACUUCUCUCAAU B gUgGuggACU UCUCUCaAU UsU B 363R-008380794-000S 1665  2 GUGGUGGACUUCUCUCAAU AuuGAGAGAAGuccAccAcUsU 234R-008380795-000A 1663  1 UCGUGGUGGACUUCUCUCA B UCG U GGUggaCuUCUCUCaUsU B 286 R-008380795-000A 1663  1 UCGUGGUGGACUUCUCUCAUGAgagaagUCCaCCaCgaUsU 238 R-008380796-000J 1663  1 UCGUGGUGGACUUCUCUCAB U CgugG U G G ACU UCUCUcA UsU B 338 R-008380796-000J 1663  1UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 240 R-008380797-000T 1663  1UCGUGGUGGACUUCUCUCA B U CgUGGUGGaCUU CuCU CAUsU B 302 R-008380797-000T1663  1 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGAUsU 260 R-008380798-000B1663  1 UCGUGGUGGACUUCUCUCA B uCGuGG UgGaCUU CUCUcA UsU B 412R-008380798-000B 1663  1 UCGUGGUGGACUUCUCUCA UGAgagaagUCCaCCaCgaUsU 242R-008418316-000S 1662 43 CUCGUGGUGGACUUCUCUC B cucGuGGuGGAcuucucucUsU B416 R-008418316-000S 1662 43 CUCGUGGUGGACUUCUCUC GAGAGAAGuccAccAcGAGUsU417 R-008418319-000T 2046 44 CCUAUGGGAGUGGGCCUCA B ccuAuGGGAGuGGGccucAUsU B 418 R-008418319-000T 2046 44 CCUAUGGGAGUGGGCCUCAUGAGGcccAcucccAuAGGUsU 419 R-008418328-000B  568 45 AAGAACUCCCUCGCCUCGCB AAGAAcucccucGccucGcUsU B 420 R-008418328-000B  568 45AAGAACUCCCUCGCCUCGC GCGAGGcGAGGGAGuucuuUsU 421 R-008418337-000K 2666 46UCUGCCGAUCCAUACUGCG B ucuGccGAuccAuAcuGcG UsU B 422 R-008418337-000K2666 46 UCUGCCGAUCCAUACUGCG CGCAGuAuGGAucGGcAGAUsU 423 R-008418340-000S1652 47 AGAGUCUAGACUCGUGGUG B AGAGucuAGAcucGuGGuG UsU B 424R-008418340-000S 1652 47 AGAGUCUAGACUCGUGGUG CACcAcGAGucuAGAcucuUsU 425R-008418343-000T  564 48 GAAGAAGAACUCCCUCGCC B GAAGAAGAAcucccucGccUsU B426 R-008418343-000T  564 48 GAAGAAGAACUCCCUCGCC GGCGAGGGAGuucuucuucUsU427 R-008418355-000C   52 49 UCAAGCCUCCAAGCUGUGCB ucAAGccuccAAGcuGuGcUsU B 428 R-008418355-000C   52 49UCAAGCCUCCAAGCUGUGC GCAcAGcuuGGAGGcuuGAUsU 429 R-008418358-000D   55 50AGCCUCCAAGCUGUGCCUU B AGccuccAAGcuGuGccuuUsU B 430 R-008418358-000D   5550 AGCCUCCAAGCUGUGCCUU AAGGcAcAGcuuGGAGGcuUsU 431 R-008418361-000K 165951 AGACUCGUGGUGGACUUCU B AGAcucGuGGuGGAcuucuUsU B 432 R-008418361-000K1659 51 AGACUCGUGGUGGACUUCU AGAAGuccAccAcGAGucuUsU 433 R-008418313-000R 564 48 GAAGAAGAACUCCCUCGCC B GAAGAAGAACUCCCUCGCCUsU B 434R-008418313-000R  564 48 GAAGAAGAACUCCCUCGCC GGCGAGGGAGUUCUUCUUCUsU 435R-008418322-000Z 1659 51 AGACUCGUGGUGGACUUCU B AGACUCGUGGUGGACUUCUUsU B436 R-008418322-000Z 1659 51 AGACUCGUGGUGGACUUCU AGAAGUCCACCACGAGUCUUsU437 R-008418325-000A 2666 46 UCUGCCGAUCCAUACUGCGB UCUGCCGAUCCAUACUGCGUsU B 438 R-008418325-000A 2666 46UCUGCCGAUCCAUACUGCG CGCAGUAUGGAUCGGCAGAUsU 439 R-008418331-000H  568 45AAGAACUCCCUCGCCUCGC B AAGAACUCCCUCGCCUCGCUsU B 440 R-008418331-000H  56845 AAGAACUCCCUCGCCUCGC GCGAGGCGAGGGAGUUCUUUsU 441 R-008418334-000J   5249 UCAAGCCUCCAAGCUGUGC B UCAAGCCUCCAAGCUGUGCUsU B 442 R-008418334-000J  52 49 UCAAGCCUCCAAGCUGUGC GCACAGCUUGGAGGCUUGAUsU 443 R-008418346-000U  55 50 AGCCUCCAAGCUGUGCCUU B AGCCUCCAAGCUGUGCCUUUsU B 444R-008418346-000U   55 50 AGCCUCCAAGCUGUGCCUU AAGGCACAGCUUGGAGGCUUsU 445R-008418349-000V 1662 43 CUCGUGGUGGACUUCUCUC B CUCGUGGUGGACUUCUCUCUsU B446 R-008418349-000V 1662 43 CUCGUGGUGGACUUCUCUC GAGAGAAGUCCACCACGAGUsU447 R-008418352-000B 2046 44 CCUAUGGGAGUGGGCCUCAB CCUAUGGGAGUGGGCCUCAUsU B 448 R-008418352-000B 2046 44CCUAUGGGAGUGGGCCUCA UGAGGCCCACUCCCAUAGGUsU 449 R-008418364-000L 1652 47AGAGUCUAGACUCGUGGUG B AGAGUCUAGACUCGUGGUGUsU B 450 R-008418364-000L 165247 AGAGUCUAGACUCGUGGUG CACCACGAGUCUAGACUCUUsU 451wherein:A, C, G, and U=ribose A, C, G or Ua, g, c and u=2′-deoxy-2′-fluoro A, G, C or UA, U, C and G=2′-O-methyl (2′-OMe) A, U, C, or GA, U, C, and G=deoxy A, U, C, or GB=inverted abasicT=thymidines=phosphorothioate linkageFurther Synthesis Steps for Commercial Preparation

Once analysis indicates that the target product purity has been achievedafter the annealing step, the material is transferred to the tangentialflow filtration (TFF) system for concentration and desalting, as opposedto doing this prior to the annealing step.

Ultrafiltration: The annealed product solution is concentrated using aTFF system containing an appropriate molecular weight cut-off membrane.Following concentration, the product solution is desalted viadiafiltration using Milli-Q water until the conductivity of the filtrateis that of water.

Lyophilization: The concentrated solution is transferred to a bottle,flash frozen and attached to a lyophilizer. The product is thenfreeze-dried to a powder. The bottle is removed from the lyophilizer andis now ready for use.

Initial Screening Protocol (96-Well Plate Transfections)

Cell Culture Preparation:

Human hepatoma cell line, HepG2, or mouse hepatoma cell line, Hepa1-6,were grown in modified Eagle's medium. All the culture media weresupplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, 100U/mL penicillin, and 1% sodium bicarbonate.

Transfection and Screening

A plasmid (pSiCheck-HepB-Partial, Ref ID: 1104) was generated where apartial HBV genome was inserted in to the pSiCheck2 plasmid (Promega,Madison, Wis.) at the XhoI/NotI sites downstream of the RenillaLuciferase gene. The Renilla Luciferase mRNA transcript is thenexpressed upstream of the HBV transcript and therefore, siNAS targetedagainst HBV genome that cause knockdown of the HBV transcript will alsoresult in knockdown of the Renilla Luciferase transcript. The level ofRenilla Luciferase transcript can be determined by directly mestuing theamount of Renilla Luciferase activity. The plasmid also expresses theFirefly Luciferase gene that is unaffected by the HBV siNAs and can beused to normalize the data to take into account any variations in theamount of plasmid transfected into cells.

The partial HBV genome consisted of 2270 nts of the adw2 genotype A HBVvirus (nts 158-2427 where EcoRI site is nt1).

Partial HBV DNA sequence (SEQ ID NO: 503):gagaacatcacatcaggatccctaggacccctgctcgtgttacaggcggggtttttcttgttgacaagaatcctcacaataccgcagagtctcgactcgtggtggacttctctcaattttctagggggatcacccgtgtgtcttggccaaaattcgcagtccccaacctccaatcactcaccaacctcctgtcctccaatttgtcctggttatcgctggatgtgtctgcggcgttttatcatattcctcttcatcctgctgctatgcctcatcttcttattggtacttctggattatcaaggtatgttgcccgtttgtcctctaattccaggaaccacaacaaccagtacgggaccctgcaaaacctgcacgactcctgctcaaggcaactctatgtttccctcatgttgctgtacaaaacctacggatggaaattgcacctgtattcccatcccatcatcttgggctttcgcaaaatacctatgggaatgggcctcagtccgtttctcatggctcagttcactagtgccatttgttcagtggttcgtagggctttcccccactgtttggctttcagttatgtggatgatgtggtattgggggccaagtctgtacaacatcttgagtccctttataccgctattaccaattttcttttgtctttgggtatacatctaaaccctaacaaaacaaagagatggggttactccctaaacttcatgggatatgtaattggaagttggggaacattaccacaggatcatattgtacaaaaacttaaacactgttttagaaaacttcctgttaataggcctattgattggaaagtatgtcaacgaattgtgggtcttttgggctttgccgctccttttacacaatgtggataccctgccttaatgcctttgtatgcatgtatacaagctaaacaggcttttactttctcgccaacttacaaggcctttctaagtaaacagtatatgaacctttaccccgttgcccggcaacggcctggtctgtgccaagtgtttgctgatgcaacccccactggctggggcttggccattggccatcagcgcatgcgcggaacctttgcggctcctctgccgatccatactgcggaactcctagcagcttgttttgctcgcagcaggtctggagcaaaactcatcgggactgataattctgtcgtcctttctcggaaatatacatcttttccatggctgctaggttgtactgccaactggattcttcgcgggacgtcctttgtttacgtcccgtcggcgctgaatcccgcggacgacccctctcggggtcgcttgggactctatcgtccccttctccgtctgccgttccagccgaccacggggcgcacctctctttacgcggtctccccgtctgtgccttctcatctgccggaccgtgtgcacttcgcttcacctctgcacgttgcatggagaccaccgtgaacgcccatcagagcctgcccaaggtcttacataagaggactcttggactctcatcaatgtcaacgaccgaccttgaagcttacttcaaagactgtgtgtttaaagactgggaagagtcgggggaggaaattaggttaaaggtttatttattaggaggctgtaggcataaattggtctgcgcaccagcaccatgcaactttttcacctctgcctaatcatctcttgtacatgtcccactgttcaagcctccaagctgtgccttgggtggctttggggcatggacattgacccctataaagaatttggagctactgtggagttactctcatttttgccttctgacttctttccttccgtccgggatctccttgataccgcctcagctctgtatcgggaagccttagagtctcctgagcattgctcacctcaccatacagcactcaggcaagccattctctgctggggggaattaatgactctagctacctgggtgggtaataatttgcaagatccagcatccagggatctagtagtcaattatgttaatactaacatgggcctaaagatcaggcaactattgtggtttcatatttcttgtcttacttttggaagagatactgtgcttgagtatttggtttctttcggagtgtggattcgcactcctcctgcctatagaccaccaaatgcccctatcttatcaacacttccggaaactactgttgttagacgaagggaccgaggtaggtcccctagaagaagaactccctcgcctcgcagacgaagatctcaatcgccgcgtcgcag

Cells were plated in all wells of tissue-culture treated, 96-well platesat a final count of 10000 cells/well in 100 μL of the appropriateculture media. The cells were cultured for overnight after plating at37° C. in the presence of 5% CO₂.

On the next day, complexes containing siNA, pSiCheck-HepB-Partialplasmid, and Lipofectamine (Invitrogen) were created as follows. Asolution of siNA and plasmid was generated containing 500 nM siNA and 30ng/ul plasmid. In parallel, a solution of Lipofectamine diluted 33-foldin OPTI-MEM was prepared. After incubation of RNAiMax/OPTI-MEM solutionat room temperature for 5 min, an equal volume of the siNA solution andthe RNAiMax solution were added together for each of the siNAs.

Mixing resulted in a solution of siNA/RNAiMax where the concentration ofsiNA was 60 nM and the plasmid was 3.6 ng/μ1. This solution wasincubated at room temperature for 20 minutes. After incubation, 20 μL ofthe solution was added to each of the relevant wells. The finalconcentration of siNA in each well was 10 nM and 0.6 ng/μ1 for theplasmid. The final volume of Lipofectamine in each well was 0.3 ul.

For 12-point dose response curve studies, the siNA series are 6-foldserial dilution starting at 30 nM or 4-fold serial dilution starting at40 nM. All transfections were set up as multiple biological replicates.

The time of incubation with the Lipofectamine-siNA complexes was 48hours and there was no change in media between transfection andharvesting for screening and dose response curve studies.

Luciferase Assays

50 μl of the Dual-Glo™ Firefly reagent (Promega, Madison, Wis.) wasadded to each well of the 96 well-plate and then the plate was incubatedat room temperature for 10 minutes. The firefly luminescence was thenread on a Wallac Envison plate reader using the Luciferase protocol. 50μl of a 1:100 dilution of the Stop & Glo substrate (Promega, Madison,Wis.) was added to each well of the 96 well-plate and then the plate wasincubated at room temperature for 10 minutes. The Renilla luminescencewas then read on a Wallac Envison plate reader using the Luciferaseprotocol.

Calculations

The expression level of the gene of interest and % inhibition of geneexpression (% KD) was calculated using a Comparative method:L_firefly=log 2(firefly luc luminosity)L_renilla=log 2(renilla luc luminosity)dL=L_firefly−L_renilladdL(log 2(foldchange))=dL_siRNA−dL_NTCRelative expression level=2^(−ddL)% KD=100×(1−2^(−ddL))

The non-targeting control siNA was, unless otherwise indicated, chosenas the value against which to calculate the percent inhibition(knockdown) of gene expression, because it is the most relevant control.

Additionally, only normalized data, which reflects the general health ofthe cell and quality of the plasmid and siNAs, was examined. This wasdone by looking at the level of two different mRNAs in the treatedcells, the first being the target mRNA and the second being thenormalizer mRNA. This is done by comparing the log 2 luminescence forrenilla luciferase (target) in each well relative to the log 2luminescence for firefly luciferase (normalizer) for the entire plate.This allowed for elimination of siNAs that might be potentially toxic tocells rather than solely knocking down the gene of interest.

All calculations of IC₅₀s were performed using R.2.9.2 software. Thedata were analyzed using the sigmoidal dose-response (variable slope)equation for simple ligand binding. In all of the calculations of thepercent inhibition (knock-down), the calculation was made relative tothe normalized level of expression of the gene of interest in thesamples treated with the non-targeting control (Ctrl siNA) unlessotherwise indicated.

Results:

The HBV siNAs were designed and synthesized as described previously.Various siNAs were screened in HepG2 or Hepa1-6 cells transfected withthe pSiCheck-HepB-Partial plasmid. The log 2(fold change) in HBV mRNAexpression levels upon treatment with various modified HBV siNAs inhuman cells is shown in Table 3a and b. Each screen was performed at 48hrs using the luciferase assay method.

TABLE 3a Primary screening data in HepG2 Cells transfected withpSiCheck-HepB-partial (n = 2), recorded as log 2(fold change) in HBVmRNA expression levels Mean log 2(fold SD log 2(fold siNA Duplex IDchange) change) R-008351268-000C 6.91 0.06 R-008351278-000V 5.40 0.02R-008351342-000A 5.18 0.16 R-008351172-000H 5.43 0.09 R-008351362-000K4.33 0.09 R-008351389-000F 4.82 0.21 R-008351306-000R 5.12 0.08R-008351372-000C 4.33 0.22 R-008351317-000S 5.07 0.06 R-008351210-000W4.79 0.08 R-008351329-000B 4.71 0.20 R-008351350-000A 4.87 0.20R-008351303-000P 4.97 0.06 R-008351229-000S 4.99 0.06 R-008351386-000E5.08 0.08 R-008351300-000N 4.61 0.24 R-008351263-000J 4.49 0.02R-008351190-000A 4.62 0.04 R-008351326-000A 4.67 0.09 R-008351260-000H4.44 0.16 R-008351195-000U 4.37 0.08 R-008351323-000Z 4.42 0.21R-008351367-000D 4.11 0.06 R-008351251-000Z 4.78 0.14 R-008351240-000Y4.51 0.28 R-008351178-000K 4.26 0.17 R-008351353-000B 4.43 0.00R-008351297-000W 4.13 0.12 R-008351175-000J 1.36 0.20 R-008351181-000S2.68 0.12 R-008351184-000T 2.45 0.02 R-008351187-000U 0.51 0.13R-008351192-000T 0.39 0.05 R-008351198-000V 0.52 0.22 R-008351201-000M2.18 0.27 R-008351204-000N 0.48 0.25 R-008351207-000P 0.90 0.02R-008351212-000N 0.45 0.26 R-008351215-000P 2.94 0.12 R-008351218-000R1.24 0.02 R-008351220-000N 0.33 0.09 R-008351223-000P 0.64 0.17R-008351226-000R 0.22 0.16 R-008351232-000Y 2.98 0.32 R-008351235-000Z0.85 0.01 R-008351237-000S 3.12 0.11 R-008351243-000Z 3.79 0.09R-008351246-000A 0.35 0.20 R-008351248-000T 0.52 0.29 R-008351254-000A3.87 0.30 R-008351257-000B 3.13 0.05 R-008351266-000K 3.84 0.13R-008351271-000J 3.04 0.18 R-008351274-000K 0.33 0.24 R-008351276-000C2.09 0.15 R-008351281-000B 3.71 0.20 R-008351284-000C 3.96 0.16R-008351287-000D 2.63 0.06 R-008351290-000K 0.47 0.27 R-008351292-000C4.11 0.17 R-008351294-000V 4.13 0.16 R-008351309-000S 1.91 0.16R-008351312-000Y 1.32 0.05 R-008351315-000Z 2.25 0.05 R-008351320-000Y0.48 0.22 R-008351332-000H 3.23 0.01 R-008351334-000A 2.09 0.60R-008351337-000B 3.65 0.15 R-008351339-000U 0.11 0.27 R-008351345-000B2.93 0.01 R-008351348-000C 0.95 0.24 R-008351356-000C 1.54 0.49R-008351359-000D 0.88 0.15 R-008351364-000C 4.01 0.10 R-008351370-000K4.17 0.04 R-008351374-000V 1.59 0.16 R-008351377-000W 1.37 0.22R-008351380-000C 1.80 0.08 R-008351383-000D 0.79 0.13 R-008351392-000M3.91 0.01 R-008351395-000N 4.03 0.05 R-008351398-000P 0.69 0.12R-008351401-000G 1.03 0.19 R-008351404-000H 2.57 0.10

TABLE 3b Primary screening data in HepG2 Cells transfected withpSiCheck-HepB-partial (n = 2), recorded as log 2(fold change) in HBVmRNA expression levels Mean log 2(fold SD log 2(fold siNA Duplex IDchange) change) R-008351268-000C 6.41 0.01 R-008351278-000V 5.26 0.05R-008351342-000A 5.01 0.13 R-008351172-000H 5.29 0.01 R-008351362-000K4.50 0.20 R-008351389-000F 4.99 0.05 R-008351306-000R 4.81 0.10R-008351372-000C 4.85 0.01 R-008351317-000S 5.45 0.04 R-008351210-000W4.71 0.17 R-008351329-000B 4.69 0.16 R-008351350-000A 4.79 0.00R-008351303-000P 4.57 0.04 R-008351229-000S 4.87 0.23 R-008351386-000E4.98 0.28 R-008351300-000N 4.48 0.06 R-008351263-000J 4.96 0.08R-008351190-000A 4.61 0.04 R-008351326-000A 4.80 0.07 R-008351260-000H4.16 0.18 R-008351195-000U 4.35 0.00 R-008351323-000Z 4.07 0.06R-008351367-000D 4.28 0.08 R-008351251-000Z 4.96 0.91 R-008351240-000Y4.63 0.01 R-008351178-000K 4.07 0.12 R-008351353-000B 4.20 0.02R-008351297-000W 4.10 0.21 R-008351175-000J 1.26 0.28 R-008351181-000S2.56 0.03 R-008351184-000T 2.57 0.25 R-008351187-000U 0.52 0.01R-008351192-000T 0.01 0.25 R-008351198-000V 0.49 0.18 R-008351201-000M2.26 0.11 R-008351204-000N 0.74 0.19 R-008351207-000P 0.58 0.34R-008351212-000N 0.18 0.26 R-008351215-000P 3.17 0.14 R-008351218-000R1.32 0.04 R-008351220-000N 0.15 0.05 R-008351223-000P 0.42 0.05R-008351226-000R 0.49 0.07 R-008351232-000Y 3.06 0.27 R-008351235-000Z0.93 0.09 R-008351237-000S 2.68 0.01 R-008351243-000Z 3.85 0.10R-008351246-000A 0.34 0.01 R-008351248-000T 0.93 0.37 R-008351254-000A4.15 0.17 R-008351257-000B 3.61 0.00 R-008351266-000K 3.87 0.25R-008351271-000J 2.67 0.26 R-008351274-000K 0.43 0.16 R-008351276-000C1.85 0.15 R-008351281-000B 3.76 0.97 R-008351284-000C 4.29 0.00R-008351287-000D 2.06 0.09 R-008351290-000K 0.11 0.14 R-008351292-000C3.99 0.01 R-008351294-000V 4.07 0.18 R-008351309-000S 1.99 0.21R-008351312-000Y 1.11 0.09 R-008351315-000Z 2.15 0.06 R-008351320-000Y0.35 0.19 R-008351332-000H 2.86 0.04 R-008351334-000A 1.71 0.01R-008351337-000B 3.59 0.01 R-008351339-000U 0.21 0.18 R-008351345-000B3.09 0.23 R-008351348-000C 0.92 0.12 R-008351356-000C 1.60 0.09R-008351359-000D 0.76 0.01 R-008351364-000C 4.37 0.05 R-008351370-000K3.99 0.19 R-008351374-000V 1.06 0.10 R-008351377-000W 1.20 0.09R-008351380-000C 1.33 0.25 R-008351383-000D 0.24 0.19 R-008351392-000M3.83 0.08 R-008351395-000N 3.58 0.01 R-008351398-000P 0.53 0.01R-008351401-000G 1.04 0.09 R-008351404-000H 2.82 0.04

A subset of siNAs from Table 3a and Table 3b having a large log 2(foldchange) in the primary screen were re-racked onto a single 96-well platein triplicate and screened again in human HepG2 cells transfected withpSiCheck_HepB-partial in order to confirm knockdown and to directlycompare siNAs that were synthesized on different plates. Table 4summarizes the data. The siNAs were ranked based on log 2(fold change)in HBV mRNA expression levels.

TABLE 4 Log2(fold change) of various siNAs tested on a re-racked plateMean log SD log siNA Duplex ID 2(fold change) 2(fold change)R-008351268-000C 6.38 0.17 R-008351278-000V 5.71 0.23 R-008351342-000A5.21 0.22 R-008351172-000H 5.00 0.11 R-008351362-000K 4.58 0.20R-008351389-000F 4.84 0.44 R-008351306-000R 5.13 0.18 R-008351372-000C4.77 0.09 R-008351317-000S 5.19 0.13 R-008351210-000W 4.68 0.09R-008351329-000B 4.71 0.08 R-008351350-000A 4.54 0.16 R-008351303-000P4.94 0.05 R-008351229-000S 4.85 0.13 R-008351386-000E 5.03 0.11R-008351300-000N 4.51 0.20 R-008351263-000J 4.74 0.28 R-008351190-000A4.50 0.15 R-008351326-000A 4.47 0.05 R-008351260-000H 4.33 0.27R-008351195-000U 4.30 0.03 R-008351323-000Z 4.30 0.08 R-008351367-000D4.23 0.06 R-008351251-000Z 4.21 0.09 R-008351240-000Y 4.14 0.22R-008351178-000K 4.11 0.08 R-008351353-000B 3.86 0.06 R-008351297-000W3.79 0.20

Select high ranking (larger log 2(fold change) value) siNAs from Table 4were further analyzed for efficacy and potency in HepG2 cellstransfected with pSiCheck-HepB-partial Plasmid. Cells were treated witha 1:6 serial dose titration of siNA in order to establish a value. ThePotency50 is the calculated siNA transfection concentration thatproduces 50% target mRNA knockdown. The results for these siNAs areshown in Table 5.

TABLE 5 Dose response data for various siNAs in HepG2-Human hepatomacell line transfected with pSiCheck-HepB-partial. Calculated maximum log2(fold change) is determined from the dose response curve. ssDRC max logPotency50 siNA Duplex ID 2(fold change) (nM) R-008351268-000C 7.00 0.02R-008351278-000V 5.59 0.11 R-008351342-000A 5.59 0.05 R-008351172-000H5.52 0.07 R-008351362-000K 5.52 0.15 R-008351389-000F 5.33 0.16R-008351306-000R 5.25 0.19 R-008351372-000C 5.21 0.09 R-008351317-000S5.12 0.11 R-008351210-000W 4.93 0.17 R-008351329-000B 4.92 0.05R-008351350-000A 4.92 0.18 R-008351303-000P 4.81 0.15 R-008351229-000S4.81 0.03 R-008351386-000E 4.71 0.34 R-008351300-000N 4.63 0.15R-008351263-000J 4.46 0.16 R-008351190-000A 4.45 0.10

Select top performing sequences identified in the previous screens,based on high ranking (larger log 2(fold change) value and low Potency₅₀values from Table 5 were then synthesized in various chemicalmodifications on both passenger and guide strand. These siNAs were thenanalyzed for efficacy and potency in Hepa1-6 cells transfected withpSiCheck-HepB-partial Plasmid. The log 2(fold change) in HBV mRNAexpression levels upon treatment with various modified HBV siNAs inhuman cells is shown in Table 6A. Each screen was performed at 48 hrsusing the luciferase assay method.

TABLE 6A Primary screening data of Lead Optimized HBV Sequences inHepa1-6 Cells transfected with pSiCheck-HepB-partial (n = 2), recordedas log 2(fold change) in HBV mRNA expression levels Mean log SD log siNADuplex ID 2(fold change) 2(fold change) R-008380633-000U 6.29 0.03R-008380783-000R 6.23 0.11 R-008380330-000N 6.19 0.06 R-008380665-000N6.18 0.15 R-008380648-000E 6.15 0.20 R-008380558-000M 6.08 0.10R-008380365-000J 5.96 0.20 R-008380406-000Y 5.96 0.10 R-008380764-000P5.95 0.02 R-008380394-000C 5.94 0.21 R-008380740-000V 5.93 0.03R-008380408-000R 5.90 0.11 R-008380576-000E 5.87 0.02 R-008380645-000D5.86 0.03 R-008380777-000H 5.85 0.18 R-008380730-000C 5.82 0.10R-008380363-000S 5.81 0.07 R-008380767-000R 5.79 0.23 R-008380359-000B5.79 0.09 R-008380763-000F 5.76 0.04 R-008380362-000H 5.75 0.19R-008380771-000F 5.72 0.09 R-008380340-000F 5.72 0.05 R-008380417-000Z5.66 0.09 R-008380377-000U 5.65 0.07 R-008380389-000D 5.63 0.19R-008380772-000P 5.58 0.03 R-008380785-000H 5.56 0.03 R-008380680-000E5.47 0.02 R-008380453-000J 5.46 0.02 R-008380750-000M 5.42 0.02R-008380776-000Z 5.41 0.30 R-008380391-000B 5.40 0.07 R-008380793-000H5.35 0.09 R-008380402-000N 5.33 0.26 R-008380516-000A 5.27 0.03R-008380492-000V 5.22 0.25 R-008380718-000M 5.21 0.02 R-008380603-000S5.20 0.04 R-008380729-000N 5.19 0.02 R-008380795-000A 5.14 0.09R-008380429-000J 5.13 0.07 R-008380760-000E 5.12 0.04 R-008380459-000L5.12 0.11 R-008380733-000D 5.06 0.22 R-008380683-000F 5.04 0.04R-008380564-000V 5.02 0.01 R-008380582-000M 5.01 0.15 R-008380418-000H4.98 0.04 R-008380540-000A 4.93 0.07 R-008380651-000L 4.91 0.12R-008380774-000G 4.89 0.01 R-008380489-000N 4.86 0.14 R-008380656-000E4.86 0.11 R-008380739-000F 4.79 0.01 R-008380462-000T 4.73 0.08R-008380324-000F 4.72 0.06 R-008380773-000Y 4.69 0.17 R-008380361-000Z4.69 0.35 R-008380798-000B 4.69 0.10 R-008380432-000R 4.66 0.16R-008380796-000J 4.63 0.03 R-008380794-000S 4.63 0.03 R-008380420-000F4.61 0.11 R-008380543-000B 4.59 0.16 R-008380642-000C 4.57 0.04R-008380731-000L 4.55 0.11 R-008380346-000H 4.50 0.02 R-008380378-000C4.47 0.29 R-008380477-000D 4.46 0.01 R-008380405-000P 4.43 0.16R-008380749-000Y 4.42 0.27 R-008380537-000U 4.41 0.12 R-008380436-000A4.40 0.19 R-008380742-000M 4.34 0.13 R-008380765-000Y 4.31 0.09R-008380353-000Z 4.29 0.10 R-008380712-000K 4.22 0.02 R-008380784-000Z4.20 0.07 R-008380379-000L 4.20 0.24 R-008380753-000N 4.19 0.09R-008380689-000H 4.15 0.06 R-008380654-000M 4.15 0.14 R-008380374-000T4.13 0.32 R-008380782-000G 4.08 0.07 R-008380480-000K 4.06 0.15R-008380766-000G 4.02 0.26 R-008380348-000A 4.01 0.21 R-008380734-000M3.97 0.09 R-008380486-000M 3.97 0.09 R-008380579-000F 3.97 0.11R-008380437-000J 3.93 0.14 R-008380781-000Y 3.91 0.10 R-008380416-000R3.88 0.29 R-008380343-000G 3.88 0.19 R-008380704-000K 3.88 0.29R-008380797-000T 3.85 0.13 R-008380736-000E 3.84 0.02 R-008380706-000C3.83 0.20 R-008380751-000W 3.83 0.17 R-008380756-000P 3.83 0.21R-008380732-000V 3.79 0.20 R-008380321-000E 3.75 0.05 R-008380755-000F3.71 0.27 R-008380528-000K 3.67 0.08 R-008380531-000S 3.64 0.20R-008380426-000H 3.60 0.02 R-008380311-000M 3.58 0.18 R-008380737-000N3.54 0.20 R-008380757-000Y 3.52 0.07 R-008380787-000A 3.46 0.08R-008380726-000M 3.46 0.35 R-008380678-000G 3.44 0.18 R-008380597-000Y3.40 0.09 R-008380686-000G 3.37 0.11 R-008380636-000V 3.31 0.05R-008380791-000R 3.27 0.24 R-008380567-000W 3.27 0.08 R-008380624-000K3.21 0.02 R-008380775-000R 3.21 0.04 R-008380315-000X 3.19 0.04R-008380407-000G 3.13 0.21 R-008380741-000D 3.10 0.20 R-008380352-000R3.09 0.07 R-008380450-000H 3.08 0.09 R-008380549-000D 3.05 0.08R-008380789-000T 3.04 0.11 R-008380746-000X 3.03 0.10 R-008380510-000Y3.00 0.04 R-008380703-000B 3.00 0.02 R-008380664-000E 2.98 0.15R-008380754-000X 2.98 0.06 R-008380759-000R 2.96 0.14 R-008380399-000W2.94 0.13 R-008380735-000W 2.92 0.13 R-008380609-000U 2.86 0.16R-008380386-000C 2.85 0.16 R-008380778-000S 2.82 0.11 R-008380428-000A2.78 0.31 R-008380483-000L 2.75 0.07 R-008380444-000A 2.73 0.13R-008380716-000V 2.69 0.16 R-008380356-000A 2.69 0.06 R-008380667-000F2.69 0.23 R-008380671-000W 2.68 0.10 R-008380705-000U 2.67 0.16R-008380410-000N 2.65 0.16 R-008380431-000G 2.61 0.07 R-008380738-000X2.60 0.06 R-008380427-000S 2.54 0.13 R-008380313-000E 2.49 0.30R-008380728-000E 2.43 0.05 R-008380792-000Z 2.40 0.02 R-008380588-000P2.40 0.17 R-008380397-000D 2.39 0.01 R-008380423-000G 2.37 0.07R-008380434-000H 2.37 0.14 R-008380316-000F 2.27 0.21 R-008380555-000L2.25 0.00 R-008380790-000G 2.23 0.09 R-008380398-000M 2.21 0.07R-008380699-000A 2.16 0.25 R-008380719-000W 2.15 0.22 R-008380335-000G2.14 0.45 R-008380618-000C 2.06 0.11 R-008380672-000E 2.02 0.02R-008380534-000T 2.02 0.00 R-008380381-000J 2.00 0.22 R-008380674-000X1.97 0.02 R-008380725-000D 1.97 0.10 R-008380770-000X 1.97 0.11R-008380369-000U 1.86 0.17 R-008380662-000M 1.86 0.07 R-008380333-000P1.78 0.21 R-008380413-000P 1.73 0.23 R-008380748-000P 1.73 0.16R-008380688-000Z 1.71 0.10 R-008380747-000F 1.70 0.18 R-008380337-000Z1.62 0.15 R-008380711-000B 1.62 0.07 R-008380522-000H 1.61 0.17R-008380308-000F 1.56 0.08 R-008380387-000L 1.54 0.03 R-008380720-000K1.50 0.07 R-008380769-000H 1.48 0.21 R-008380404-000F 1.46 0.04R-008380786-000S 1.46 0.03 R-008380630-000T 1.44 0.05 R-008380727-000W1.41 0.19 R-008380779-000A 1.38 0.04 R-008380594-000X 1.34 0.05R-008380438-000T 1.32 0.06 R-008380456-000K 1.32 0.00 R-008380768-000Z1.30 0.00 R-008380600-000R 1.28 0.00 R-008380762-000X 1.25 0.18R-008380370-000H 1.19 0.17 R-008380349-000J 1.19 0.36 R-008380390-000T1.19 0.31 R-008380447-000B 1.13 0.06 R-008380666-000X 1.12 0.25R-008380639-000W 1.11 0.03 R-008380546-000C 1.05 0.07 R-008380685-000Y1.04 0.02 R-008380698-000S 1.04 0.09 R-008380414-000Y 1.03 0.10R-008380401-000E 1.02 0.24

A subset of siNAs from Table 6a having a large log 2(fold change) in theprimary screen were re-racked onto a single 96-well plate in triplicateand screened again in murine Hepa1-6 cells transfected withpSiCheck_HepB-partial in order to confirm knockdown and to directlycompare siNAs that were synthesized on different plates. Table 6B.summarizes the data. The siNAs were ranked based on log 2(fold change)in HBV mRNA expression levels.

TABLE 6B Log2(fold change) of various siNAs tested on a re-racked plateMean log 2(fold SD log 2(fold siNA Duplex ID change) change)R-008380665-000N 6.79 0.09 R-008380648-000E 6.53 0.09 R-008380633-000U6.45 0.05 R-008380783-000R 6.45 0.06 R-008380408-000R 6.36 0.05R-008380645-000D 6.34 0.04 R-008380767-000R 6.32 0.01 R-008380764-000P6.26 0.18 R-008380406-000Y 6.23 0.04 R-008380740-000V 6.21 0.14R-008380730-000C 6.19 0.06 R-008380362-000H 6.17 0.06 R-008380340-000F6.12 0.10 R-008380558-000M 6.11 0.09 R-008380389-000D 6.10 0.22R-008380363-000S 6.09 0.06 R-008380777-000H 6.08 0.07 R-008380772-000P6.02 0.03 R-008380576-000E 6.00 0.05 R-008380771-000F 5.99 0.03R-008380359-000B 5.99 0.12 R-008380377-000U 5.97 0.06 R-008380763-000F5.94 0.11 R-008380680-000E 5.90 0.01 R-008380776-000Z 5.86 0.04R-008380793-000H 5.86 0.04 R-008380417-000Z 5.85 0.07 R-008380785-000H5.83 0.04 R-008380750-000M 5.63 0.02 R-008380420-000F 5.10 0.09R-008380782-000G 4.47 0.31 R-008380753-000N 4.45 0.15 R-008380689-000H4.42 0.04 R-008380755-000F 4.36 0.13 R-008380736-000E 4.35 0.05R-008380346-000H 4.35 0.02 R-008380756-000P 4.32 0.01 R-008380734-000M4.21 0.09 R-008380781-000Y 4.17 0.03 R-008380737-000N 4.03 0.26R-008380757-000Y 3.97 0.06 R-008380791-000R 3.89 0.03 R-008380528-000K3.78 0.06 R-008380787-000A 3.65 0.13 R-008380686-000G 3.63 0.16R-008380754-000X 3.54 0.08 R-008380597-000Y 3.49 0.07 R-008380321-000E3.47 0.09 R-008380567-000W 3.43 0.05 R-008380624-000K 3.27 0.08R-008380789-000T 3.20 0.09 R-008380746-000X 3.18 0.03 R-008380778-000S3.13 0.10 R-008380735-000W 3.08 0.09 R-008380399-000W 2.82 0.02R-008380410-000N 2.60 0.08 R-008380311-000M 1.24 0.07 R-008380353-000Z1.11 0.03 R-008380348-000A 0.75 0.04 R-008380315-000X 0.69 0.06

Select high ranking (larger log 2(fold change) value) siNAs from Table6b were further analyzed for efficacy and potency in Hepa1-6 cellstransfected with pSiCheck-HepB-partial Plasmid. Cells were treated witha 1:6 serial dose titration of siNA in order to establish a value. ThePotency50 is the calculated siNA transfection concentration thatproduces 50% target mRNA knockdown. The results for these siNAs areshown in Table 6c.

TABLE 6c Dose response data for various siNAs in Hepal-6 murine hepatomacell line transfected with pSiCheck-HepB-partial. Calculated maximum log2(fold change) is determined from the dose response curve. ssDRC max logPotency50 siNA Duplex ID 2(fold change) (nM) R-008380648-000E 7.78 0.104R-008380783-000R 7.01 0.023 R-008380665-000N 6.97 0.008 R-008380645-000D6.92 0.032 R-008380408-000R 6.80 0.016 R-008380633-000U 6.79 0.012R-008380767-000R 6.72 0.028 R-008380730-000C 6.58 0.038 R-008380777-000H6.57 0.020 R-008380740-000V 6.55 0.019 R-008380558-000M 6.45 0.014R-008380406-000Y 6.32 0.006 R-008380764-000P 6.27 0.030 R-008380772-000P6.26 0.024 R-008380389-000D 6.22 0.021 R-008380362-000H 6.17 0.014R-008380363-000S 5.87 0.009 R-008380340-000F 5.76 0.016 R-008380689-000H5.30 0.036 R-008380756-000P 5.00 0.186 R-008380736-000E 4.96 0.076R-008380757-000Y 4.88 0.216 R-008380753-000N 4.87 0.184 R-008380737-000N4.69 0.142 R-008380734-000M 4.65 0.226 R-008380791-000R 4.37 0.101

Example 2: Determining In Vitro Serum Stability of siNAs

siNAs are reconstituted as 50 μM to 100 μM stock solution with H₂O andadded to human serum pre-warmed to 37° C. to a final concentration of 20m/mL. The mixture is then incubated at 37° C. for 0, 1 and 2 hours. Atthe end of each time point, the reactions are stopped by mixing withequal volume of Phenomenex Lysis-Loading Buffer. Oligonucleotides arepurified in 96-well format by Phenomenex Solid Phase Extraction andlyophilized until dry with Labconco Triad Lyo-00417. The lyophilizedsamples are reconstituted in 150 μL of 1 mM EDTA prepared withRNase-free H₂O. The sample solutions re then diluted 5 fold with 1 mMEDTA for liquid chromatography/mass spectrometry (LC/MS) analysis onThermoFisher Orbitrap. Serum metabolites of the siNAs were determinedbased on the measured molecular weights.

Example 3: Testing of Cytokine Induction

To assess immunostimulative effects of various siNAs of the inventionloaded in lipid nanoparticles (e.g.,DLinDMA/Cholesterol/S-PEG-C-DMA/DSPC in a 40/48/2/10 ratio), C57Bl/6mice are dosed with a single 3mpk dose of LNP formulated siNAs throughtail vein injection. Serum or plasma samples are collected at 3 and 24hours post-dose. The cytokine and chemokine levels in these samples ismeasured with the SearchLight IR Cytokine Array from Aushon Biosciencesaccording to the manufacturer's instruction. The cytokines andchemokines measured are IL-1α, IL-1β, IL-6, KC, IL-10, IFNγ, TNF, GMCSF,MIP-1β, MCP-1/JE, and RANTES.

Example 4: Efficacy Studies in Mouse

In vivo efficacy studies are conducted in SCID mice with humanizedlivers that are subsequently inject with HBV. Transgenic mice withdiseased livers are injected with human primary hepatocytes that allowsrepopulation of the mouse liver with up to 70% human hepatocytes. Miceare then inoculated with human HBV isolates. Infected mice are dosed IVvia tail vein injections with LNP encapsulated siNAs or vehicle controlusing a single 3mpk dose. Effect of siNA on viral load is determinedtaking serum sample at various time points post-dosing and thenmeasuring the level of circulating HBV genomes in the serum by qPCR.

Example 5: Pharmacodynamic Study in Non-Human Primates

HBV-infected chimpanzees are dosed with a single 2.5mpk dose of siNAloaded lipid nanoparticles through intravenous infusion. To monitor HBVvirus load, serum samples are taken at various time points pre- andpost-dose and the level of HBV genomes are determined via qPCR. Allprocedures adhere to the regulations outlined in the USDA Animal WelfareAct (9 CFR, Parts 1, 2 and 3) and the conditions specified in The Guidefor Care and Use of Laboratory Animals (ILAR publication, 1996, NationalAcademy Press).

Example 6: siNAs LNP Formulations

A. General LNP Process Description for DLinDMA Formulations:

The lipid nanoparticles are prepared by an impinging jet process. Theparticles are formed by mixing lipids dissolved in alcohol with siNAdissolved in a citrate buffer. The mixing ratio of lipids to siNA istargeted at 45-55% lipid and 65-45% siNA. The lipid solution contains acationic lipid, a helper lipid (cholesterol), PEG (e.g. PEG-C-DMA,PEG-DMG) lipid, and DSPC at a concentration of 5-15 mg/mL with a targetof 9-12 mg/mL in an alcohol (for example ethanol). The ratio of thelipids has a mole percent range of 25-98 for the cationic lipid with atarget of 35-65, the helper lipid has a mole percent range from 0-75with a target of 30-50, the PEG lipid has a mole percent range from 1-15with a target of 1-6, and the DSPC has a mole percent range of 0-15 witha target of 0-12. The siNA solution contains one or more siNA sequencesat a concentration range from 0.3 to 1 0.0 mg/mL with a target of0.3-0.9 mg/mL in a sodium citrate buffered salt solution with pH in therange of 3.5-5. The two liquids are heated to a temperature in the rangeof 15-40° C., targeting 30-40° C., and then mixed in an impinging jetmixer instantly forming the LNP. The teeID has a range from 0.25 to 1.0mm and a total flow rate from 10-600 mL/minute The combination of flowrate and tubing ID has the effect of controlling the particle size ofthe LNPs between 30 and 200 nm. The solution is then mixed with abuffered solution at a higher pH with a mixing ratio in the range of 1:1to 1:3 vol:vol but targeting 1:2 vol:vol. This buffered solution is at atemperature in the range of 15-40° C., targeting 30-40° C. The mixedLNPs are held from 30 minutes to 2 hrs prior to an anion exchangefiltration step. The temperature during incubating is in the range of15-40° C., targeting 30-40° C. After incubating, the solution arefiltered through a 0.8 um filter containing an anion exchange separationstep. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and aflow rate from 10 to 2000 mL/minute The LNPs are concentrated anddiafiltered via an ultrafiltration process where the alcohol is removedand the citrate buffer is exchanged for the final buffer solution suchas phosphate buffered saline. The ultrafiltration process uses atangential flow filtration format (TFF). This process uses a membranenominal molecular weight cutoff range from 30-500 KD. The membraneformat is hollow fiber or flat sheet cassette. The TFF processes withthe proper molecular weight cutoff retains the LNP in the retentate andthe filtrate or permeate contains the alcohol; citrate buffer; and finalbuffer wastes. The TFF process is a multiple step process with aninitial concentration to a siNA concentration of 1-3 mg/mL. Followingconcentration, the LNPs solution is diafiltered against the final bufferfor 10-20 volumes to remove the alcohol and perform buffer exchange. Thematerial is then concentrated an additional 1-3 fold. The final steps ofthe LNP process are to sterile filter the concentrated LNP solution andvial the product.

Analytical Procedure:

1) siNA Concentration

The siNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with a liner gradient from 0-15 min and a flow rate of1 ml/minute. The siNA amount is determined by comparing to the siNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siNA and total siNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission is measured at 530 nm. The siNA amount isdetermined by comparing to an siNA standard curve.Encapsulation rate=(1−free siNA/total siNA)×100%

3) Particle Size and Polydispersity

RDVs containing 1 μg siNA are diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples is measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity is measured with He—Ne laser at 25° C. with a scattering angleof 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changes from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with a flow rate of 1ml/minute. The individual lipid concentration is determined by comparingto the standard curve with all the lipid components in the RDVs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

B. General Formulation Procedure for CLinDMA/Cholesterol/PEG-DMG at aRatio of 71.9:20.2:7.9.

siNA solutions were prepared by dissolving siNAs in 25 mM citrate buffer(pH 4.0) at a concentration of 0.8 mg/mL. Lipid solutions were preparedby dissolving a mixture of 2S-Octyl-ClinDMA, cholesterol and PEG-DMG ata ratio of 71.9:20.2:7.9 in absolute ethanol at a concentration of about10 mg/mL. Equal volume of siNA and lipid solutions were delivered withtwo syringe pumps at the same flow rates to a mixing T connector. Theresulting milky mixture was collected in a sterile bottle. This mixturewas then diluted slowly with an equal volume of citrate buffer, andfiltered through a size exclusion hollow fiber cartridge to remove anyfree siNA in the mixture. Ultra filtration against citrate buffer (pH4.0) was employed to remove ethanol (test stick from ALCO screen), andagainst PBS (pH 7.4) to exchange buffer. The final LNP was obtained byconcentrating to a desired volume and sterile filtered through a 0.2 mmfilter. The obtained LNPs were characterized in term of particle size,alcohol content, total lipid content, nucleic acid encapsulated, andtotal nucleic acid concentration.

C. General LNP Preparation for Various Formulations in Table 10

siNA nanoparticle suspensions in Table 10 were prepared by dissolvingsiNAs and/or carrier molecules in 20 mM sodium citrate buffer (pH 5.0)at a concentration of about 0.40 mg/mL. Lipid solutions were prepared bydissolving a mixture of cationic lipid (e.g.,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, see structurein Table 11), DSPC, Cholesterol, and PEG-DMG (ratios shown in Table 10)in absolute ethanol at a concentration of about 8 mg/mL. The nitrogen tophosphate ratio was approximated to 6:1.

Nearly equal volumes of siNA/carrier and lipid solutions were deliveredwith two FPLC pumps at the same flow rates to a mixing T connector. Aback pressure valve was used to adjust to the desired particle size. Theresulting milky mixture was collected in a sterile glass bottle. Thismixture was then diluted with an equal volume of citrate buffer,followed by equal volume of PBS (pH 7.4), and filtered through anion-exchange membrane to remove any free siNA/carrier in the mixture.Ultra filtration against PBS (7.4)) was employed to remove ethanol andto exchange buffer. The final LNP was obtained by concentrating to thedesired volume and sterile filtered through a 0.2 μm filter. Theobtained LNPs were characterized in term of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

LNP Manufacture Process

In a non-limiting example, LNPs were prepared in bulk as follows. Theprocess consisted of (1) preparing a lipid solution; (2) preparing ansiNA/carrier solution; (3) mixing/particle formation; (4) incubation;(5) dilution; (6) ultrafiltration and concentration.

1. Preparation of Lipid Solution

2 L glass reagent bottles and measuring cylinders were depyrogenated.The lipids were warmed to room temperature. Into the glass reagentbottle was transferred 8.0 g of(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine with a pipetteand 1.2 g of DSPC, 3.5 g of Cholesterol, 0.9 g of PEG-DMG were added. Tothe mixture is added 1 L of ethanol. The reagent bottle was placed inheated water bath, at a temperature not exceeding 50° C. The lipidsuspension was stirred with a stir bar. A thermocouple probe was putinto the suspension through one neck of the round bottom flask with asealed adapter. The suspension was heated at 30-40° C. until it becameclear. The solution was allowed to cool to room temperature.

2. Preparation of siNA/Carrier Solution

Into a sterile container (Corning storage bottle) was weighed 0.4 gtimes the water correction factor (approximately 1.2) of siNA powder.The siNA was transferred to a depyrogenated 2 L glass reagent bottle.The weighing container was rinsed 3× with citrate buffer (20 mM, pH 5.0)and the rinses were placed into the 2 L glass bottle, QS with citratebuffer to 1 L. The concentration of the siNA solution was determinedwith a UV spectrometer using the following procedure. 20 μL was removedfrom the solution, diluted 50 times to 1000 μL, and the UV readingrecorded at A260 nm after blanking with citrate buffer. This wasrepeated. Note, if the readings for the two samples are consistent, anaverage can be taken and the concentration calculated based on theextinction coefficients of the siNAs. If the final concentration is outof the range of 0.40±0.01 mg/mL, the concentration can be adjusted byadding more siNA/carrier powder, or adding more citrate buffer. Thisprocess can be repeated for the second siNA, if applicable

When the siNA/carrier solution comprised a single siNA duplex instead ofa cocktail of two or more siNA duplexes and/or carriers, then thesiNA/carrier was dissolved in 20 mM citrate buffer (pH 5.0) to give afinal concentration of 0.4 mg/mL.

The lipid and ethanol solutions were then sterile filtered through aPall Acropak 20 0.8/0.2 μm sterile filter PN 12203 into a depyrogenatedglass vessel using a Master Flex Peristaltic Pump Model 7520-40 toprovide a sterile starting material for the encapsulation process. Thefiltration process was run at an 80 mL scale with a membrane area of 20cm². The flow rate was 280 mL/minute. This process can be scaled byincreasing the tubing diameter and the filtration area.

3. Particle Formation—Mixing Step

Using a two-barrel syringe driven pump (Harvard 33 Twin Syringe), thesterile lipid/ethanol solution and the sterile siNA/carrier orsiNA/carrier cocktail/citrate buffer (20 mM citrate buffer, pH 5.0)solutions were mixed in a 0.5 mm ID T-mixer (Mixing Stage I) at equal,or nearly equal, flow rates. The resulting outlet LNP suspensioncontained 40-50 vol % ethanol. To obtain a 45 vol % ethanol outletsuspension, the sterile lipid/ethanol and the sterile siNA/carrier orsiNA/carrier cocktail/citrate buffer solutions were mixed at flow ratesof 54 mL/min and 66 mL/min, respectively, such that the total flow rateof the mixing outlet is 120 mL/min.

4. Dilution

The outlet stream of Mixing Stage I was fed directly into a 4 mm IDT-mixer (Mixing Stage II), where it was diluted with a buffered solutionat higher pH (20 mM sodium citrate, 300 mM sodium chloride, pH 6.0) at aratio of 1:1 vol:vol %. This buffered solution was at a temperature inthe range of 30-40° C., and was delivered to the 4 mm T-mixer via aperistaltic pump (Cole Parmer MasterFlex L/S 600 RPM) at a flow rate of120 mL/min.

The outlet stream of Mixing Stage II was fed directly into a 6 mm IDT-mixer (Mixing Stage III), where it was diluted with a bufferedsolution at higher pH (PBS, pH 7.4) at a ratio of 1:1 vol:vol %. Thisbuffered solution was at a temperature in the range of 15-25° C., andwas delivered to the 6 mm T-mixer via peristaltic pump (Cole ParmerMasterFlex L/S 600 RPM) at a flow rate of 240 mL/min.

5. Incubation and Free siNA Removal

The outlet stream of Mixing Stage III was held after mixing for 30minute incubation. The incubation was conducted at temperature of 35-40°C. and the in-process suspension was protected from light. Followingincubation, free (un-encapsulated) siNA was removed via anion exchangewith Mustang Q chromatography filters (capsules). Prior to use, thechromatography filters were pre-treated sequentially with flushes of INNaOH, 1M NaCl, and a final solution of 12.5 vol % ethanol in PBS. The pHof the final flush was checked to ensure pH<8. The incubated LNP streamwas then filtered via Mustang Q filters via peristaltic pump (ColeParmer MasterFlex L/S 600 RPM) at flow rate of approximately 100 mL/min.The filtered stream was received into a sterile glass container forultrafiltration and concentration as follows.

6. Ultrafiltration, Concentration and Sterile Filtration

The ultrafiltration process is a timed process and the flow rates mustbe monitored carefully. This is a two step process; the first is aconcentration step taking the diluted material and concentratingapproximately 8-fold, to a concentration of approximately 0.3-0.6 mg/mLsiNA.

In the first step, a ring-stand with a ultrafiltration membrane 100 kDaPES (Spectrum Labs) installed was attached to a peristaltic pump(Spectrum KrosFloII System). 9.2 L of sterile distilled water was addedto the reservoir; 3 L was drained to waste and the remainder was drainedthrough permeate to waste. 5.3 L of 0.25 N sodium hydroxide was added tothe reservoir with 1.5 L drained to waste and 3.1 L drained throughpermeate to waste. The remaining sodium hydroxide was held in the systemfor sanitization (at least 10 minutes), and then the pump was drained.9.2 L of 70 (v/v) % isopropyl alcohol was added to the reservoir with1.5 L drained to waste and the remainder drained through permeate towaste. 6 L of conditioning buffer (12.5% ethanol in phosphate bufferedsaline) was added with 1.5 L drained to waste and the remainder drainedthough the permeate until the waste was of neutral pH (7-8). A membraneflux value was recorded, and the pump was then drained.

The diluted LNP solution was placed into the reservoir to the 1.1 Lmark. The pump was turned on at 2.3 L/min. After 5 minutes ofrecirculation, the permeate pump was turned on at 62.5 mL/min and theliquid level was constant at approximately 950 mL in the reservoir. Thediluted LNP solution was concentrated from 9.8 L to 1.1 L in 140minutes, and the pump was paused when all the diluted LNP solution hasbeen transferred to the reservoir.

The second step was a diafiltration step exchanging the ethanol/aqueousbuffer to phosphate buffered saline. During this step, approximately10-20 diafiltration volumes of phosphate buffered saline were used.Following diafiltration, a second concentration was undertaken toconcentrate the LNP suspension 3-fold to approximately 1-1.5 mg/mLsiRNA. The concentrated suspension was collected into sterile, plasticPETG bottles. The final suspension was then filtered sequentially viaPall 0.45 um PES and Pall 0.2 um PES filters for terminal sterilizationprior to vial filling.

The obtained LNPs were characterized in terms of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

D. Synthesis of Novel Cationic Lipids

Synthesis of the novel cationic lipids is a linear process starting fromlipid acid (i). Coupling to N,O-dimethyl hydroxylamine gives the Weinrebamide ii. Grignard addition generates ketone iii. Titanium mediatedreductive amination gives final products of type iv.

Synthesis of the single carbon homologated cationic lipids v is a linearprocess starting from lipid ketone (iii). Conversion of the ketone tothe nitrile (iv) is accomplished via treatment with TOSMIC and potassiumtert-butoxide. Reduction of the nitrile to the primary amine followed byreductive amination provides final cationic lipids v.

Synthesis of two carbon homologated cationic lipids viii is a linearprocess starting from lipid ketone (iii). Conversion of the ketone tothe α,β-unsaturated amide vi is accomplished under Peterson conditions.Conjugate reduction of the α,β-unsaturation is performed usingLS-Selectride to give amide vii. Reduction of the amide with lithiumaluminum hydride provides final cationic lipids viii.

Cyclopropyl containing lipids are prepared according to General Scheme4. Unsaturated Weinreb amides ii are subjected to Simmons-Smithcyclopropanation conditions to give cyclopropyl containing Weinrebamides ix. These are carried on to final products as outlined in GeneralSchemes 1-3.

Synthesis of allylic amine cationic lipids xv is a linear processstarting with aldehyde x. Addition of t-butyl aceate generates β-hydroxyester xi. Conversion of the hydroxyl functionality to a fluoro groupfollowed by acid treatment generates β-fluoro acid xii. Conversion ofthe acid to the Weinreb amide followed by Grignard addition gives theβ-fluoro ketone xiv. Reductive amination results in simultaneouselimination to generate the desired allylic amine xv.

11,14-Eicosadienoic acid, (11Z,14Z)— (50 g, 162 mmol),N,O-Dimethylhydroxylamine hydrochloride (31.6 g, 324 mmol), HOAt (44.1g, 324 mmol), Et₃N (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) weremixed in DCM (810 mL) and stirred overnight at ambient temperature.Reaction was then washed 5×700 mL water, then washed 1×600 mL 1 M NaOH,dried with sodium sulfate, filtered through celite and evaporated toobtain 53.06 g (93%) 11,14-eicosadienamide, N-methoxy-N-methyl-,(11Z,14Z) as a clear golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),3.68 (s, 3H), 3.18 (s, 3H), 2.77 (m, 2H), 2.41 (t, J=7 Hz, 2H), 2.05 (m,4H), 1.63 (m, 2H), 1.40-1.26 (m, 18H), 0.89 (t, J=7 Hz, 3H).

11,14-eicosadienamide, N-methoxy-N-methyl-, (11Z,14Z)— 1 (4 g, 11.38mmol) was dissolved in dry THF (50.0 ml) in a 250 mL flask then 1 Mnonylmagnesium bromide (22.76 nil, 22.76 mmol) was added under nitrogenat ambient temperature. After 10 min, the reaction was slowly quenchedwith excess sat. aq NH₄Cl. The reaction was washed into a separatoryfunnel with hexane and water, shaken, the lower aqueous layer discarded,the upper layer dried with sodium sulfate, filtered, and evaporated togive crude ketone as a golden oil. To the above crude ketone was addeddimethylamine (2 M in THF) (14.22 ml, 28.4 mmol) followed by Ti(O-i-Pr)₄(6.67 ml, 22.76 mmol) and let stir overnight. The next day, added EtOH(50 ml) followed by NaBH₄ (0.646 g, 17.07 mmol). After 5 min ofstirring, directly injected entire reaction onto a 40 g silica columnthat was in line with a 330 g silica column. Eluted 10 min 100% DCM,then 30 min 0-15% MeOH/DCM, collected 20,23-nonacosadien-10-amine,N,N-dimethyl-, (20Z,23Z) (1) (2.45 g, 5.47 mmol, 48.1% yield) as afaintly golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H), 2.78 (m,2H), 2.23 (m, 1H), 2.21 (s, 6H), 2.05 (m, 4H), 1.45-1.16 (m, 38H), 0.89(m, 6H). HRMS calcd for C31H61N, 448.4877, found 448.4872.

Compounds 2-30 are novel cationic lipids and were prepared according tothe General Scheme 1 above. Com- pound Structure HRMS  2

calcd C28H56N 406.4407, found 406.4405.  3

calcd C27H54N 392.4251, found 392.4250.  4

calcd C24H48N 350.3781, found 350.3770.  5

calcd C23H46N 336.3625, found 336.3613.  6

calcd C25H50N 364.3938, found 364.3941.  7

calcd C26H52N 378.4094, found 378.4081.  8

calcd C29H58N 420.4564, found 420.4562.  9

calcd C26H52N 378.4094, found 378.4089. 10

calcd C25H50N 364.3938, found 364.3931. 11

calcd C30H60N 434.4720, found 434.4717. 12

calcd C29H58N 420.4564, found 420.4561. 13

calcd C28H56N 406.4407, found 406.4404. 14

calcd C27H54N 392.4251, found 392.4245. 15

calcd C33H66N 476.5190, found 476.5196. 16

calcd C32H64N 462.5033, found 462.5045. 17

calcd C29H59N 422.4720, found 422.4726. 18

calcd C28H57N 408.4564, found 408.4570. 19

calcd C30H59N 434.4720, found 434.4729. 20

calcd C29H61N 424.4877, found 424.4875. 21

calcd C32H64N 462.5033, found 462.5023. 22

calcd C33H64N 474.5033, found 474.5033. 23

calcd C29H60N 422.4720, found 422.4716. 24

calcd C29H60N 422.4720, found 422.4718. 25

calcd C31H64N 450.5033, found 450.5031. 26

calcd C31H64N 450.5033, found 450.5034. 27

calcd C35H72N 506.5659, found 506.5635. 28

calcd C31H64N 450.5033, found 450.5037. 29

calcd C33H68N 478.5346, found 478.5358. 30

calcd C27H56N 394.4407, found 394.4407.

(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (Compound 31)

A solution of keton iii (4.0 g, 9.55 mmol), TOSMIC (2.4 g, 12.4 mmol) indimethoxyethane (45 mL) was cooled to 0° C. and treated with potassiumtert-butoxide (19.1 mmol, 19.1 mL of a 1M solution in tBuOH). After 90minutes, the reaction was partitioned between hexanes and water. Theorganics were washed with water, dried over sodium sulfate, filtered andevaporated in vacuo. This material was purified by flash chromatography(0-5% EtOAc/hexanes) to give desired product (containing ˜20% of s.m.).This mixture was carried into next step as is. LC/MS (M+H)=430.6.

Lithium aluminum hydride (23.9 mmol, 23.9 mL of a 1M solution in THF)was added directly to nitrile iv (3.42 g, 8 mmol) at ambient temperatureand the reaction was stirred for 20 minutes. The reaction was dilutedwith 100 mL THF, cooled to 0° C. and carefully quenched with sodiumsulfate decahydrate solution. The solids were filtered off and washedwith THF. The filtrate was evaporated in vacuo and carried directly intonext reaction crude. LC/MS (M+H)=434.6.

A solution of primary amine (3.45 g, 6.2 mmol) in dichloroethane (100mL) was treated with formaldehyde (1.6 mL, 21.7 mmol) followed by sodiumtriacetoxyborohydride (6.6 g, 31 mmol). After 5 minutes, the reactionwas partitioned between dichloromethane and 1N NaOH. The organics weredried over sodium sulfate, filtered and evaporated in vacuo. The crudemixture was purified by reverse phase preparative chromatography (C8column) to provide(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine. HRMS calc'd462.5033, found 462.5026. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H), 2.78(2H, t, J=5.6 Hz), 2.18 (s, 6H), 2.05 (m, 6H), 1.3 (m, 39H), 0.89 (m,6H).

(13Z,16Z)—N,N-dimethyl-3-nonyldocosa- 13,16-dien-1-amine (Compound 32)

The silyl amide Peterson reagent (3.1 g, 16.7 mmol) was dissolved in THF(35 mL) and cooled to −63° C. To this solution was added nBuLi (16.7mmol, 6.7 mL of a 2.5M solution). The reaction was warmed to ambienttemperature for 30 minutes. The ketone (5.0 g, 11.9 mmol) was dissolvedin THF (25 mL) in a second flask. The Peterson reagent was transferredto the ketone solution at −60° C. The reaction was warmed to −40° C. for1 hour, then warmed to 0° C. for 30 minutes. The reaction was quenchedwith sodium bicarbonate, diluted with additional water and partitionedbetween water/hexanes. The organics were washed with brine, dried oversodium sulfate, filtered and evaporated in vacuo. Purification by flashchromatography (0-40% MTBE/hexanes) gave α,β-unsatured amide vi. ¹H NMR(400 MHz, CDCl₃) δ 5.75 (s, 1H), 5.36 (m, 4H), 3.01 (s, 3H), 2.99 (s,3H), 2.78 (t, 2H), 2.28 (t, 2H), 2.05 (m, 6H), 1.35 (m, 34H), 0.89 (m,6H).

α,β-unsatured amide vi (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1mL of a 1M solution) were combined in a sealed tube and heated to 60° C.for 24 hours. The reaction was cooled to ambient temperature andpartitioned between ammonium chloride solution and heptane. The organicswere dried over sodium sulfate, filtered and evaporated in vacuo to giveamide vii. This intermediate was carried directly into next reactioncrude.

To a solution of amide vii (2.85 g, 5.8 mmol) was added lithium aluminumhydride (8.7 mmol, 8.7 mL of a 1M solution). The reaction was stirred atambient temperature for 10 minutes then quenched by slow addition ofsodium sulfate decahydrate solution. The solids were filtered and washedwith THF and the filtrate evaporated in vacuo. The crude mixture waspurified by reverse phase preparative chromatography (C8 column) toprovide (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine(Compound 32) as an oil. HRMS (M+H) calc'd 476.5190, found 476.5189. ¹HNMR (400 MHz, CDCl₃) δ 5.37 (m, 4H), 2.78 (t, 2H), 2.42 (m, 8H), 2.05(q, 4H), 1.28 (m, 41H), 0.89 (m, 6H).

N,N-dimethyl-1-(2-octylcyclopropyl)heptadecan-8-amine (Compound 33)

To a solution of oleic acid (1 g, 3.5 mmol) in DCM (500 mL) cooled to 0°C. was added CDI (0.63 g, 3.9 mmol). The reaction was warmed to ambienttemperature for 30 minutes before cooling to 0° C. and treating firstwith triethylamine (0.39 g, 3.9 mmol) and then dimethyl hydroxylaminehydrochloride (0.38 g, 3.9 mmol). After 1 hour the reaction waspartitioned between water and heptane. The organics were dried overmagnesium sulfate, filtered and evaporate in vacuo to give crude Weinrebamide ii which was carried directly into next reaction.

A solution of diethylzinc (70.3 mmol, 70.3 mL of a 1M solution) indichloromethane (130 mL) was cooled to −1° C. and treated dropwise withTFA (8.0 g, 70.3 mmol). After 30 minutes, diiodomethane (18.8 g, 70.3mmol) was added and this was aged for 30 minutes in the ice bath. Tothis solution was added Weinreb amide ii (7.6 g, 23.4 mmol). Thereaction was warmed to ambient temperature and stirred for 1 hour. Thereaction was quenched with ammonium chloride solution (100 mL) andorganic layer partitioned off, washed with 10% sodium thio sulfate,dried over magnesium sulfate, filtered and evaporated in vacuo.Purification was flash chromatography (0-30% MTBE/heptane) gave desiredproduct ix. ¹H NMR (400 MHz, CDCl₃) δ 3.72 (s, 3H), 3.22 (s, 3H), 2.48(t, 2H), 1.65 (m, 2H), 1.39 (m, 22H), 1.18 (m, 2H), 0.91 (t, 3H), 0.68(m, 2H), 0.59 (m, 1H), −0.32 (m, 1H).

Conversion of Weinreb amide ix to Compound 33 was carried out in amanner analogous to that described for Compound 1 above (nonyl Grignardaddition followed by reductive amination). LC/MS (M+H)=436.6. ¹H NMR(400 MHz, CDCl₃) δ 2.25 (s, 6H), 1.30 (m, 45H), 0.91 (m, 6H), 0.68 (m,2H), 0.59 (m, 1H), −0.31 (m, 1H).

Compounds 34-43 are novel cationic lipids and were prepared according toGeneral Schemes 1-4 above.

Compound Structure HRMS 34

calcd C30H62N 436.4877, found 436.4872. 35

calcd C32H66N 464.5190, found 464.5186. 36

calcd C34H70N 492.5503, found 492.5496. 37

calcd C33H66N 476.5190, found 476.5174. 38

calcd C29H60N 422.4720, found 422.4701. 39

calcd C30H62N 436.4877, found 436.4880. 40

calcd C32H66N 464.5190, found 464.5199. 41

calcd C30H62N 436.4877, found 436.4877. 42

calcd C30H62N 436.4877, found 436.4875. 43

LC/MS (M + H) 408.6.

(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine (Compound 44)

To a solution of LDA (95 mmol, 47.5 mL of a 2M solution) in THF (127 mL)cooled to −78° C. was added t-butyl acetate. The reaction was stirredfor 15 minutes followed by addition of aldehyde x. The reaction wasimmediately quenched with ammonium chloride solution, warmed to ambienttemperature and partitioned between water/pentane. The organics weredried over sodium sulfate, filtered and evaporated in vacuo. LC/MS(M+H-tBu)=325.4.

Hydroxy ketone xi (7 g, 18.4 mmol) was dissolved in dichloromethane (150mL) and cooled to 0° C. and treated with deoxofluor (7.3 g, 33.1 mmol).The reaction was warmed to ambient temperature with stirring for 16hours followed by quenching with sodium bicarbonate solution. Thereaction was partitioned and the organics dried over sodium sulfate,filtered and evaporate in vacuo. Flash column chromatography (0-5% ethylacetate/hexanes) gave the □-fluoro ester.

Fluoro ester intermediate (6 g, 15.6 mmol) in dichloromethane wastreated with hydrogen chloride (157 mmol, 39.2 mL of a 4M solution indioxane) and the reaction was stirred at ambient temperature for 16hours. The reaction was evaporated in vacuo to give desired β-fluoroacid xii. LC/MS (M+H)=327.3.

Fluoro carboxylic acid xii (5.1 g, 15.7 mmol), EDC (6.0 g, 31.4 mmol),N,O-dimethylhydroxylamine hydrochloride (3.1 g, 31.4 mmol),trimethylamine (4.0 g, 39.2 mmol), and HOAt (4.3 g, 31.4 mmol) werecombined in DCM (78 mL) and stirred at ambient temperature for 16 hours.The reaction was partitioned between water/DCM and the organics werewashed with water (3×) and NaOH solution (lx), dried over sodiumsulfate, filtered and evaporated in vacuo. Crude material was purifiedby reverse phase preparative chromatography to give desired Weinrebamide xiii. LC/MS (M+H)=370.4.

A solution of Weinreb amide xiii (4.3 g, 11.7 mmol) in THF (50 mL) wastreated with nonylmagnesium bromide (23.4 mmol, 23.4 mL of a 1Msolution) at ambient temperature. The reaction was quenched withammonium chloride solution after 1 hour and partitioned between waterand pentane. The organics were dried over sodium sulfate, filtered andevaporated in vacuo. This material was carried into next step crude.

Ketone xiv (5.1 g, 11.7 mmol) was treated with dimethylamine (29.3 mmol,14.7 mL of a 2M solution in THF) and titanium(IV) isopropoxide (6.7 g,23.5 mmol) and the reaction was stirred at ambient temperature for 16hours. To the reaction mixture was added ethanol (50 mL) followed bysodium borohydride (0.67 g, 17.6 mmol). The reaction was loaded directlyonto a silica column and purified by flash chromatography (0-15%MeOH/DCM). The material required a second purification by preparativereverse phase chromatography to give(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine. HRMS calc'd446.4720, found 446.4724. ¹H NMR (400 MHz, CDCl₃) δ 5.48 (m, 1H), 5.37(m, 4H), 5.23 (m, 1H), 2.78 (t, 2H), 2.58 (m, 1H), 2.22 (s, 6H), 2.04(m, 6H), 1.56 (m, 1H), 1.30 (m, 31H), 0.89 (m, 6H).

Compound 45 is DLinKC2DMA as described in Nature Biotechnology, 2010,28, 172-176, WO 2010/042877 A1, WO 2010/048536 A2, WO 2010/088537 A2,and WO 2009/127060 A1.

Compound 46 is MC3 as described in WO 2010/054401, and WO 2010/144740A1.

E. Lipid Nanoparticle Compositions

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiNA molecules of the invention:

Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;

Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;

Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10;

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein, as presently representative ofpreferred embodiments, are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

TABLE 7 HBV Accession Numbers X02763 - SEQ ID NO: 504 Hepatitis b virusgenome (serotype adw2) X02763.1 GI: 59418 X69798 - SEQ ID NO: 505Hepatitis B virus, subtype adw4 genes X69798.1 GI: 59422 X65259 - SEQ IDNO: 506 Hepatitis B virus (ayw,patient E) genes PreS1, PreS2, PreC, C, Xand polymerase X65259.1 GI: 59439 X04615 - SEQ ID NO: 507 Hepatitis Bvirus genome, subtype ayr X04615.1 GI: 59585 D00329 - SEQ ID NO: 508Hepatitis B virus subtype ADW genomic DNA, complete genome, clone:pJDW233 D00329.1 GI: 221497 M32138 - SEQ ID NO: 509 Hepatitis B virusvariant HBV-alphal, complete genome M32138.1 GI: 329667 X75657 - SEQ IDNO: 510 Human hepatitis virus (genotype E, Bas) preS1, preS2, S, C, X,antigens, core antigen, X protein and polymerase X75657.1 GI: 452617M12906 - SEQ ID NO: 511 Hepatitis B virus subtype adr, complete genome.M12906.1 GI: 474959 X85254 - SEQ ID NO: 512 Hepatitis B virus genome(PreS1, PreS2, S, PreC, C, X genes and polymerase) X85254.1 GI: 736003X51970 - SEQ ID NO: 513 Hepatitis B virus (HBV 991) complete genome.X51970.1 GI: 1155012 AB014381 - SEQ ID NO: 514 Hepatitis B virus genomicDNA, complete sequence, isolate 22Y04HCC AB014381.1 GI: 3582357AF100309 - SEQ ID NO: 515 Hepatitis B virus strain 56, complete genomeAF100309.1 GI: 4323201 AF090842 - SEQ ID NO: 516 Hepatitis B virusstrain G5.27295, complete genome AF090842.1 GI: 5114084 AB033554 - SEQID NO: 517 Hepatitis B virus DNA, complete genome, isolate: RTB299AB033554.1 GI: 6063442 AB032431 - SEQ ID NO: 518 Hepatitis B virusgenomic DNA, complete sequence, isolate: HBV/E-Ch195 AB032431.1 GI:6691492 AF160501 - SEQ ID NO: 519 Hepatitis B virus strain IG29227,complete genome AF160501.1 GI: 6983934 AB036910 - SEQ ID NO: 520Hepatitis B virus (genotype F) genomic DNA, complete genome, isolate:VNZ8251 AB036910.1 GI: 11191875 AF223965 - SEQ ID NO: 521 Hepatitis Bvirus strain C-1858 isolate sa16, complete genome AF223965.1 GI:12247041 AB064310 - SEQ ID NO: 522 Hepatitis B virus DNA, completegenome, clone: USG769 AB064310.1 GI: 18146661 AF405706 - SEQ ID NO: 523Hepatitis B virus isolate 235/01, complete genome AF405706.1 GI:19849032 AY090454 - SEQ ID NO: 524 Hepatitis B virus strain 1853Nic,complete genome AY090454.1 GI: 22135696 AY090457 - SEQ ID NO: 525Hepatitis B virus strain 2928Nic, complete genome AY090457.1 GI:22135711 AY090460 - SEQ ID NO: 526 Hepatitis B virus strain LAS2523,complete genome AY090460.1 GI: 22135726

TABLE 8 Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine purine caps p =S Strand “Stab 00” Ribo Ribo TT at 3′- S/AS ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoro Ribo — 1at 3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′- — Usually Sends “Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′ and 3′-— Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O-Methyl 2′-O- 5′and 3′- Usually S Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′-Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro*2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl*“Stab 29” 2′-fluoro* 2′-O- 1 at 3′-end S/AS Methyl* “Stab 30” 2′-fluoro*2′-O- S/AS Methyl* “Stab 31” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab32” 2′-fluoro 2′-O- S/AS Methyl “Stab 33” 2′-fluoro 2′-deoxy* 5′ and 3′-— Usually S ends “Stab 34” 2′-fluoro 2′-O- 5′ and 3′- Usually S Methyl*ends “Stab 35” 2′-fluoro*† 2′-O- Usually AS Methyl*† “Stab 36”2′-fluoro*† 2′-O- Usually AS Methyl*† “Stab04H” 2′-fluoro‡ Ribo‡ 5′ and3′- 1 at 3′-end Usually S ends “Stab06C” 2′-O-Methyl‡ Ribo‡ 5′ and 3′-Usually S ends “Stab07H” 2′-fluoro‡ 2′-deoxy‡ 5′ and 3′- 1 at 3′-endUsually S ends “Stab07mU” 2′-fluoro‡ 2′-deoxy‡ 5′ and 3′- Usually S ends“Stab09H” Ribo‡ Ribo‡ 5′ and 3′- 1 at 3′-end Usually S ends “Stab16C”Ribo‡ 2′-O- 5′ and 3′- Usually S Methyl‡ ends “Stab16H” Ribo‡ 2′-O- 5′and 3′- 1 at 3′-end Usually S Methyl‡ ends “Stab18C” 2′-fluoro‡ 2′-O- 5′and 3′- Usually S Methyl‡ ends “Stab18H” 2′-fluoro‡ 2′-O- 5′ and 3′- 1at 3′-end Usually S Methyl‡ ends “Stab52H” 2′-O-Methyl‡ Ribo‡ 5′ and 3′-1 at 3′-end Usually S ends “Stab05C” 2′-fluoro‡ Ribo‡ Usually AS“Stab05N” 2′-fluoro‡ Ribo‡ 1 at 3′-end Usually AS “Stab10C” Ribo‡ Ribo‡Usually AS “Stab10N” Ribo‡ Ribo‡ 1 at 3′-end Usually AS “Stab35G*”2′-fluoro‡ 2′-O- Usually AS Methyl‡ “Stab35N*” 2′-fluoro‡ 2′-O- 1 at3′-end Usually AS Methyl‡ “Stab35rev*” 2′-O-Methyl‡ 2′-fluoro‡ UsuallyAS “Stab50*” Ribo‡ 2′-O- Usually AS Methyl‡ “Stab53*” 2′-O-Methyl‡ Ribo‡Usually AS “Stab53N*” 2′-O-Methyl‡ Ribo‡ 1 at 3′-end Usually AS Stab54Ribo‡ 2′-fluoro‡ Usually AS CAP = any terminal cap, see for example FIG.6. All Stab chemistries can be used in combination with each other forduplexes of the invention(e.g., as combinations of sense and antisensestrand chemistries), or alternately can be used in isolation, e.g., forsingle stranded nucleic acid molecules of the invention. All Stabchemistries can comprise 3′-overhang nucleotides having 2′-O-alkyl,2′-deoxy-2′-fluoro, 2′-deoxy, LNA or other modified nucleotides ornon-nucleotides. All Stab chemistries typically comprise about 19-21nucleotides, but can vary as described herein. All Stab chemistries canalso include a single ribonucleotide in the sense or passenger strand atthe 11^(th) base paired position of the double-stranded nucleic acidduplex as determined from the 5′-end of the antisense or guide strand.All Stab chemistries can also have in place of the Stab designationabove a 2′-deoxy-2′-fluoro modification at position 14 from the 5′ endof the antisense strand regardless of whether it is a purine orpyrimidine at that position. All Stab chemistries of the antisensestrand presented above can have a thymidine in place of a 2′-deoxyuridine at position 1, 2, and/or 3 from the 5′ end of the antisensestrand. S = sense strand. AS = antisense strand *Stab 23 has a singleribonucleotide adjacent to 3′-CAP. *Stab 24 and Stab 28 have a singleribonucleotide at 5′-terminus. *Stab 25, Stab 26, Stab 27, Stab 35, Stab35G*, Stab 35N*, Stab 35rev*, Stab 36, Stab 50*, Stab53*, Stab 53N*, andStab 54 have three ribonucleotides at 5′-terminus. *Stab 29, Stab 30,Stab 31, Stab 33, and Stab 34 any purine at first three nucleotidepositions from 5′-terminus are ribonucleotides. p = phosphorothioatelinkage. †Stab 35 has 2′-O-methyl U at 3′-overhangs and threeribonucleotides at 5′-terminus. †Stab 36 has 2′-O-methyl overhangs thatare complementary to the target sequence. (naturally occurringoverhangs) and three ribonucleotides at 5′-terminus. ‡Stab 04H, Stab06C, Stabl07H, Stab07mU, Stab09H, Stab16C, Stab 16H, Stab18C, Stab 18H,Stab 52H, Stab 05C, Stab05N, Stab10C, Stab10N, Stab35G*, Stab35N*,Stab35N*, Stab35rev*, Stab 50*, Stab 53*, Stab 53N*, Stab 54 have two2′-O-methyl U 3′-overhangs. Stab35G*, Stab 35N*, Stab35rev*, Stab50*,Stab53*, and Stab53N* do not allow for a 2′-O-methyl modification atposition 14 of the guide strand as determined from the 5′-end.

TABLE 9 Wait Time* Wait Time* Wait Time* Reagent Equivalents Amount DNA2′-O-methyl RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL  45 sec  2.5 min  7.5 min S-Ethyl 23.8 238μL  45 sec  2.5 min  7.5 min Tetrazole Acetic 100 233 μL  5 sec  5 sec 5 sec Anhydride N-HBVhyl 186 233 μL  5 sec  5 sec  5 sec Imidazole TCA176  2.3 mL  21 sec  21 sec  21 sec Iodine 11.2  1.7 mL  45 sec  45 sec 45 sec Beaucage 12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA6.67 mL NA NA NA B. 0.2 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 15  31 μL 45 sec 233 sec 465 sec S-Ethyl 38.7  31 μL 45sec  233 min 465 sec Tetrazole Acetic 655 124 μL  5 sec  5 sec  5 secAnhydride N-HBVhyl 1245 124 μL  5 sec  5 sec  5 sec Imidazole TCA 700732 μL  10 sec  10 sec  10 sec Iodine 20.6 244 μL  15 sec  15 sec  15sec Beaucage 7.7 232 μL 100 sec 300 sec 300 sec Acetonitrile NA 2.64 mLNA NA NA Equivalents: Amount: DNA/2′-O- DNA/2′-O- Wait Time* Wait Time*Wait Time* Reagent methyl/Ribo methyl/Ribo DNA 2′-O-methyl Ribo C. 0.2μmol Synthesis Cycle 96 well Instrument Phosphoramidites 22/33/6640/60/120 μL  60 sec 180 sec 360 sec S-Ethyl 70/105/210 40/60/120 μL  60sec  180 min 360 sec Tetrazole Acetic 265/265/265 50/50/50 μL  10 sec 10 sec  10 sec Anhydride N-HBVhyl 502/502/502 50/50/50 μL  10 sec  10sec  10 sec Imidazole TCA 238/475/475 250/500/500 μL  15 sec  15 sec  15sec Iodine 6.8/6.8/6.8 80/80/80 μL  30 sec  30 sec  30 sec Beaucage34/51/51 80/120/120 100 sec 200 sec 200 sec Acetonitrile NA1150/1150/1150 NA NA NA μL *Wait time does not include contact timeduring delivery. Tandem synthesis utilizes double coupling of linkermolecule

TABLE 10 Composition of Select Lipid Nanoparticle Formulations LNP siNAN/P Identifier Lipid Components and Molar Ratios Duplex LNP-1 CompoundCholesterol DSPC PEG- R-008380648- 6 32 (30%) (10%) DMG 000E (50%) (2%)LNP-2 Compound Cholesterol DSPC PEG- R-008380783- 6 32 (30%) (10%) DMG000R (50%) (2%) LNP-3 Compound Cholesterol DSPC PEG- R-008380665- 6 32(30%) (10%) DMG 000N (50%) (2%) LNP-4 Compound Cholesterol DSPC PEG-R-008380645- 6 32 (30%) (10%) DMG 000D (50%) (2%) LNP-5 CompoundCholesterol DSPC PEG- R-008380408- 6 32 (30%) (10%) DMG 000R (50%) (2%)LNP-6 Compound Cholesterol DSPC PEG- R-008380633- 6 32 (30%) (10%) DMG000U (50%) (2%) LNP-7 Compound Cholesterol DSPC PEG- R-008380767- 6 32(30%) (10%) DMG 000R (50%) (2%) LNP-8 Compound Cholesterol DSPC PEG-R-008380730- 6 32 (30%) (10%) DMG 000C (50%) (2%) LNP-9 CompoundCholesterol DSPC PEG- R-008380777- 6 32 (30%) (10%) DMG 000H (50%) (2%)LNP-10 Compound Cholesterol DSPC PEG- R-008380740- 6 32 (30%) (10%) DMG000V (50%) (2%) LNP-11 Compound Cholesterol DSPC PEG- R-008380558- 6 32(30%) (10%) DMG 000M (50%) (2%) LNP-12 Compound Cholesterol DSPC PEG-R-008380406- 6 32 (30%) (10%) DMG 000Y (50%)6 (2%) LNP-13 CompoundCholesterol DSPC PEG- R-008380764- 6 32 (30%) (10%) DMG 000P (50%) (2%)LNP-14 Compound Cholesterol DSPC PEG- R-008380772- 6 32 (30%) (10%) DMG000P (50%) (2%) LNP-15 Compound Cholesterol DSPC PEG- R-008380389- 6 32(30%) (10%) DMG 000D (50%) (2%) LNP-16 Compound Cholesterol DSPC PEG-R-008380362- 6 32 (30%) (10%) DMG 000H (50%) (2%) LNP-17 CompoundCholesterol DSPC PEG- R-008380363- 6 32 (30%) (10%) DMG 000S (50%) (2%)LNP-18 Compound Cholesterol DSPC PEG- R-008380340- 6 32 (30%) (10%) DMG000F (50%) (2%) LNP-19 Compound Cholesterol DSPC PEG- R-008380689- 6 32(30%) (10%) DMG 000H (50%) (2%) LNP-20 Compound Cholesterol DSPC PEG-R-008380756- 6 32 (30%) (10%) DMG 000P (50%) (2%) LNP-21 CompoundCholesterol DSPC PEG- R-008380736- 6 32 (30%) (10%) DMG 000E (50%) (2%)LNP-22 Compound Cholesterol DSPC PEG- R-008380757- 6 32 (30%) (10%) DMG000Y (50%) (2%) LNP-23 Compound Cholesterol DSPC PEG- R-008380753- 6 32(30%) (10%) DMG 000N (50%) (2%) LNP-24 Compound Cholesterol DSPC PEG-R-008380737- 6 32 (30%) (10%) DMG 000N (50%) (2%) LNP-25 CompoundCholesterol DSPC PEG- R-008380734- 6 32 (30%) (10%) DMG 000M (50%) (2%)LNP-26 Compound Cholesterol DSPC PEG- R-008380791- 6 32 (30%) (10%) DMG000R (50%) (2%) N/P ratio = Nitrogen:Phosphorous ratio between cationiclipid and nucleic acid

TABLE 11 Chemical Structures of Lipids in Formulations of Table 10 LipidChemical Structure Compound 32     Cholesterol     DSPC     PEG-DMG

What we claim is:
 1. A double-stranded short interfering nucleic acid(siNA) molecule comprising a sense strand comprising the nucleotidesequence 5′-B GCCgAUCCAUACUGCGGAAUsU B-3′ (SEQ ID NO:398) and anantisense strand comprising the nucleotide sequence5′-UUCCGCAGUAUGGAUCGGCUsU-3′ (SEQ ID NO:248), wherein B is an invertedabasic moiety, bolded nucleotides comprise 2′-O-methyl modifications,lower case nucleotides comprise 2′-fluoro modifications, italicizednucleotides are deoxynucleotides, and s is a phosphorothioate linkage.2. A composition comprising the double-stranded short interferingnucleic acid (siNA) according to claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 3. A composition comprising: (a) thedouble-stranded short interfering nucleic acid (siNA) of claim 1; (b) acationic lipid; (c) cholesterol; (d) DSPC; and (e) PEG-DMG.
 4. Acomposition comprising: (a) the double-stranded short interferingnucleic acid (siNA) of claim 1; (b) (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13, 16-dien-1-amine; (c) cholesterol;(d) DSPC; and (e) PEG-DMG.
 5. The composition according to claim 4,wherein the (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,cholesterol, DSPC, and PEG-DMG have a molar ratio of 50:30:10:2respectively.
 6. A composition comprising the composition according toclaim 5 and a pharmaceutically acceptable carrier or diluent.
 7. Amethod of treating a human subject suffering from a condition which ismediated by the action, or by loss of action, of Hepatitis B Virus(HBV), which comprises administering to said subject an effective amountof the double-stranded short interfering nucleic acid (siNA) molecule ofclaim
 1. 8. The method according to claim 6, wherein the condition isHBV infection.
 9. The method according to claim 6, wherein the conditionis hepatocellular carcinoma.
 10. A kit comprising the double-strandedshort interfering nucleic acid (siNA) according to claim 1.